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Biological removal of methanethiol from gas and water

streams by using Thiobacillus thioparus:investigation of biodegradability and optimization of sulfur production

Journal: Environmental Technology

Manuscript ID: OA-TENT-2013-0577.R3

Manuscript Type: Original Article

Date Submitted by the Author: 20-Dec-2013

Complete List of Authors: Badr, Kiumars; Environmental Research Center in Petroleum and

Petrochemical Industries, Shiraz University, bahmani, mahmoud; Environmental Research Center in Petroleum and Petrochemical Industries, Shiraz University Jahanmiri, Abdolhossein; shiraz university, Mowla, Dariush; shiraz university, ; Environmental Research Center in Petroleum and Petrochemical Industries, Shiraz University

Keywords: Methanethiol removal, Biodegradability, Biological oxidation, Elemental sulfur, Thiobacillus thioparus

URL: http:/mc.manuscriptcentral.com/tent

Environmental Technology

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Biological removal of methanethiol from gas and water streams by using

Thiobacillus thioparus:investigation of biodegradability and optimization of

sulfur production

Kiumars Badr a, Mahmoud Bahmani

a, Abdolhossein Jahanmiri

b, Dariush Mowla

a, b, *

a Environmental Research Center in Petroleum and Petrochemical Industrial, School of Chemical Engineering,

Shiraz University, Shiraz, Iran b School of Chemical and Petroleum Engineering, Shiraz University, Shiraz, Iran

Abstract

The present work mainly deals with biological oxidation which was tested using the bacterium

Thiobacillus thioparus in semi-batch bioreactor systems to evaluate the removal efficiencies and

optimal conditions for the biodegradation of methanethiol (MT) in order to treat the natural gas

and refinery output streams. The efficiency of this method is analyzed by evaluating the

concentration of MT in a bioreactor. The effect of operational parameters such as initial

concentration of MT, pH, temperature, dissolved oxygen, initial concentration of bacteria and

reaction time on the degradation of MT were studied. In this process, MT is converted into

elemental sulfur particles as an intermediate in the oxidation process of MT to sulfate. The

obtained results showed that the highest degradation rate occurred during the first 300 minutes of

reaction time. The optimal conditions of the different initial MT concentration with 0.3 to 0.6

bacteria OD, dissolved oxygen of 0.5 ppm, acidic pH value of 6.2 and temperature of 30°C are

obtained. Acidic pH and oxygen-limiting conditions were applied to obtain 80 to 85 %

selectivity for elemental sulfur formation in products. Under the optimal conditions, and for the

highest (8.51mM) and the lowest (0.53mM) concentration of MT, the biological removal were

about 89% and 94% respectively.

Keywords: Methanethiol removal; Biodegradability; Biological oxidation; Elemental sulfur;

Thiobacillus thioparus.

* Corresponding author. E-mail: [email protected] (Dariush Mowla).

Tel.: +98-711-2303071

Fax: +98-711- 6473180

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1. Introduction

Mercaptans (thiols) are highly reactive sulfur-containing species well known for their

disagreeable odors. The most volatile of thiols is methanethiol (MT, CH3SH), which is one of the

natural sources of sulfur could be emitted into the atmosphere [1-3]. It is a colorless gas with a

smell like rotten cabbage. MT is commonly present in both natural gas and refinery streams.

Examples of various gas and refinery streams containing MT and hydrogen sulfide are given in

Table 1.

They have to be removed mainly for three reasons:

(a) Both MT and hydrogen sulfide are acid compounds and therefore they can cause serious

corrosion problems,

(b) They have an offensive odor and a low odor threshold,

(c) Most of them are highly toxic.

The maximum permissible concentration of gaseous MT in the work place is 5.0 ppm (threshold

limit value-time weighted average concentration, TLV-TWA) and 0.5 ppm as defined by

American Conference of Governmental Industrial Hygienists (ACGIH). In addition,

Occupational Safety and Health Administration (OSHA) defined the MT concentration to 10

ppm as permissible exposure limit time weighted average concentration (PEL TWA) [4, 5]. At a

higher concentrations MT becomes very toxic and may causes harmful health effects.

Table 1.

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Due to high chemical requirements, energy and disposal costs, the chemical and physical

removal processes of mercaptan are expensive. For example, there are two main chemical

processes for removal of MT: Merox processes [13] and Merichem processes [14] which in these

methods, MT is not removed from the fuel, but rather transformed into disulfides which are toxic

and flammable compounds.

Hence after both of the traditional treatments which have mentioned above, the sulfur content in

the fuel is not reduced by only these treatments [15]. To overcome disadvantages of

physiochemical process for mercaptan removal (e.g., high pressure, high temperature, need for

special chemicals and usually generate an additional problem in the disposition of the exhausted

reagents [16]), the use of biological treatment is an interesting alternative because of low costs,

operational simplicity and intrinsically clean technologies [17-19].

Biological treatment uses several species of bacteria to convert MT into a non-toxic form

materials such as sulfate or sulfur particles. There are different types of bioreactors, which could

be used for biological MT removal, the more common types are: bioscrubber, biotrickling filter

and biofilter.

An example of VRSC (Volatile Reduced Sulfur Compounds) gases treatment by biotrickling

filter, is the research of Rukojärvi et al. [20] who is obtained removal capacities of 48, 37 and 3

gS·m-3

·h-1

for H2S, DMS and MT, respectively, using a biotrickling filter inoculated with a

microbial consortium. Hartikainen et al. [21] reached elimination capacities of 6, 7 and 4 gS·m-

3·h

-1 for H2S, DMS and MT, respectively applying a non inoculated fibrous peat as support.

However, it has been reported that biofilters inoculated with pure cultures, mainly bacteria from

the Thiobacillus, Acidithiobacillus and Hyphomicrobium genera, depending on the biofilm

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generation conditions have a lower start-up time and a more stable operation over the time [22,

23].

In the two mentioned processes, the gas containing MT passes through a moist, packed bed of

particles, which are coated by microorganism. Because of direct contact of gas- bacteria in

biotrickling filter and biofilter systems, there is an increment in mass transfer resistance from gas

phase to liquid phase which causes mass transfer limitation and also these are appropriate for low

mercaptan capacity. In these processes, sulfate is produced with no elemental sulfur [24].

In the case of hydrogen sulfide and volatile organic sulfur compounds (VOSCs) removal from

natural gas and refinery streams, bioscrubber is more suitable than two previous processes.

Mesa et al. [25] described a bioscrubber system which could be applied into a system by a

combination of chemical and biological processes for H2S removal from biogas. H2S removal

can be achieved by absorption in a ferric sulfate solution and producing ferrous sulfate and

elemental sulfur. Ferric sulfate could be regenerated by biological oxidation using

Acidithiobacillus ferrooxidans.

Benschop et al. [26] obtained a full scale plant located northeast of Brooks, Alberta, Canada used

Shell-Paques® process for natural gas desulfurization. H2S is removed from a gaseous stream by

absorption into scrubbing liquid. The hydrogen sulfide containing scrubbing liquid is treated in

the bioreactor where it is mostly converted biologically to elemental sulfur.

Roosta et al.[27] investigated biological sulfide removal in a batch bioreactor. In this process,

hydrogen sulfide is converted into elemental sulfur particles as an intermediate in the oxidation

of hydrogen sulfide to sulfate. The main product is elemental sulfur at low dissolved oxygen.

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In the first step the gaseous stream reacts with a concentrated solution of sodium hydroxide (14

wt% NaOH) in which MT are dissolved as the corresponding sodium salts according to:

OHSNaCHNaOHSHCH aqg 2)(3)(3 +→+ (1)

The first step takes place in an extraction column, where MT are washed by a counter current of

caustic stream and the treated gas is passed overhead. The treated gas now free of MT, could be

delivered directly to the customers. The loaded scrubbing liquid or contaminated wastewater

with MT, sends to the bioreactor.

A review on bacteria of the sulfur cycle was discussed by Tang et al. [28]. In their research, for

MT reduction, aerobic microorganisms are used. In the aerobic bioreactor, MT is biologically

oxidized according to the following equations [29]:

−−++→+ OHCOSOSCH 3

25

2

0

23 (2)

+−−++→+ HCOSOOSCH 33 2

2

423 (3)

+−−++→+ HCOOSOSCH 62

272 2

2

3223 (4)

Although the formation of sulfate and thiosulfate consumes more energy than the formation of

elemental sulfur however, it is possible to obtain a selectivity between 80 and 85 percent for the

formation of elemental sulfur by applying oxygen-limiting conditions and decreasing the pH to

the extent of acidic pH [30, 31]. There is a mediated reaction for sulfate production which can

carry out in an appropriate conditions (Eq. (5)):

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+−−+→++ HSOOOHS 2

42

0

23 (5)

In appropriate temperatureand presence of enough oxygen, the non-biological reaction occurs

which is written below (Eq. (6)) [32]:

OHSSCHCHOSHCH 23323 212 +→+ (6)

The previous investigations show clearly that biological removal processes can be applied

effectively to remove MT pollution in gas. However, only a few studies have been done directly

on removal hazardous MT material effectively by low temperature and cost, high speed and

neutral pH and also produces just sulfur selectively instead of other hazardous materials [33].The

previous researches have generally checked degradation of only about 0.2 mM [34] and didn’t

mentioned the type of produced materials and also a few operational parameters have been

studied. Therefore, more detailed experimental investigations are required for better

understanding the removal efficiency of this very harmful component.

In this study, we present the results of bioreactor experiments on MT removal. Biodegradation of

MT is assessed by evaluating the effects of parameters as well as production of elemental sulfur

selectivity in a so called semi-batch bioreactor.

2. Materials and method

2.1. Strain and media

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In this study, Thiobacillus thioparus (DSMZ 5368) is used as sulfur oxidizing bacteria for

producing elemental sulfur and sulfate as the products in the oxidation of MT. The medium for

growth is shown in Table 2 [35]. Each compound was purchased in the highest purity that was

commercially available.

After addition of all compounds, the pH of the medium was 7.1 ± 0.1 at 30 ◦C. Then the strain

(100 cm3 of the medium culture containing bacteria with an OD600 of about 0.5) added to a flask

containing 2000 cm3 of the medium. After that, the medium kept in a shaker incubator with 150

rpm and temperature about 30 ˚C for five days to obtain enough concentration of bacteria.

Table 2.

2.2. Experimental set-up and procedure

A reactor with total volume of 3.8 L operated under semi-batch conditions, as shown in Figure 1

constructed. During each experiment, the temperature, dissolved oxygen, Initial MT

concentration and pH were controlled. The reactor was placed in a water bath to control the

temperature within 0.5 ˚C of the set point (30 ˚C). The pH was controlled using 1N HCl and 1N

NaOH solutions between 5.6 and 8.2. The dissolved oxygen (DO) was changed between 0.5 and

6 ppm and controlled using nitrogen and oxygen injection, it means that when their

concentrations were low or completely finish, system were charged them again through closing

and opening manual valve. The free thiosulfate and bromocresol purple main medium was the

original inoculum of this bioreactor (Table 1). The bioreactor was charged with 2000 cm3 of the

medium and inoculated with centrifuged biomass (bacteria OD = 0.3-0.6). After temperature

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stabilization, feeding of MT to the bioreactor was started. MT solution was prepared by solving

sodium methyl mercaptide (21%w) in deionized water. The concentration of prepared MT

solution was 500 mM. The concentration of this solution was standardized based on mercuric

acetate and N,N-dimethyl- p-phenylenediamine described by Moore using a spectrophotometer

(Zeiss) at 500 nm [36].

The reactor was equipped with sensors for temperature, pH (Metrohm LL-Unitrode Pt 1000),

dissolved oxygen (AZ 8403) and oxidation reduction potential (ORP Ag/AgCl electrode AZ

8501) electrode. ORP electrode is a tool in order to measure the tendency of a chemical species

to acquire electrons and thereby be reduced. Reduction potential is measured in millivolts. A

diffuser was placed in the floor of bioreactor for scattering the circulating gas. The flow rate of

the circulating gas was enough (15 Lmin-1

) for mixing the contents of the close bioreactor. The

gas was circulated by an air pump (ACO-308). Three sampling instruments were prepared on the

head of the bioreactor. The concentrations of dimethyle disulfide (DMDS) were determined

indirectly by spectrophotometry at a wavelength of 285 nm [37]. At this wavelength, MT did not

show any significant absorbance at all concentrations tested compared to the medium solution.

The total concentrations of MT were determined based on mercuric acetate and N,N-dimethyl- p-

phenylenediamine described by Moore using a spectrophotometer at 500 nm [36].The

concentration of sulfate was determined via the turbidimetry method at 420 nm [38]. While the

thiosulfate concentration was determined via methylene blue method at 760 nm [39]. The

concentration of sulfur particles was determined based on toluene described by Roosta et al.

using spectrophotometer at 290 nm [27].

An UV/VIS spectrophotometer (Optima, SP 3000 plus) was used for measurement of all

components.

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The synthetic substrate is made by dissolving MT in the liquid feed and in the ranges applicable

in the oil and gas refineries streams. For identification of sulfides volatilized from liquid feed, a

volume of 3 L of MT (6 mM) was stirred for 12 h, in the bioreactor under experimental

conditions (pH=6, T=30 ˚C). After this time, spectrophotometric analyses confirmed the

negligibility of the MT volatilization amount. Some key parameters have been checked to

determine the optimum conditions for MT removal. In the first section of the experiments, the

optimal conditions in terms of temperature, pH and dissolved oxygen are obtained for removal

the most part of MT as well as production of elemental sulfur. In the second section, in order to

determine the amount of removal efficiency and sulfur production, different initial

concentrations of MT were used and variations of MT concentration were measured versus time

at achieved optimal conditions.

Figure 1.

3. Results and discussion

3.1. Effects of temperature and pH

As it was mentioned before, for bacteria growth, they should be placed at incubator in

temperature about 30 ˚C. But the temperature may vary from the optimum value in industrial

conditions. The temperature was varied from 25˚C to 35˚C whereas other parameters were

almost kept constant.

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Variations of uneliminated MT with temperature is illustrated in Figure 2(a) which reveals that

the best performance for MT removal could be obtained at T=30˚C.

The optimal pH range for Thiobacillus thioparus is between 6 and 8. However, it can grow in

higher and lower range of pH values. The effect of pH on the uneliminated MT concentration or

in fact on bacteria activity is illustrated in Figure 2(b). As it is obvious, as pH increases,

uneliminated MT is increased, because the activity of bacteria has been decreased. This fact is

correct for pH values lower than 6.

At pH higher than 8.2 and lower than 6 the activity was reduced sharply, however in the pH

between 6 and 8.2 change of activity was low. Furthermore, at high and very low pH values, OD

of bacteria is decreased. The reasons for concentration reduction of bacteria could be proved by

two facts: first, the bacteria cannot grow at high or low pH and second, the activity reduction

causes more accumulation of MT in the bioreactor and high concentration of MT is toxic for the

bacteria [40, 41].

Because of oxygen lacking, a large amount of MT was converted to sulfur particles according to

Eq. 2. Thiosulfate is an undesired product in biological MT removal which is produced during

sulfur production according to Eq. 4. The effect of pH on thiosulfate production is demonestrated

in Figure 2(c). The result shows that the production of thiosulfate decreases with the pHs

reduction. Based on reactions 2 and 4 it is found out, OH- is produced along with production of

elemental sulfur which leads to alkaline conditions of reactor (Eq. 2).Beside it, H+ as well as

thiosulfate are produced which cause acidic conditions in reactor (Eq. 4). According to Le

Chatelier's principle, as pH in reactor decreases, reaction 2 is reinforced and reaction 4 is

prevented. Furthermore , since the rate constant of reaction 4 at pH=6 is low in comparison with

that of reactions 2 and 3, production of thiosulfate is negligible [42, 43].

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Figure 2.

In addition, the effect of bacteria concentration on uneliminated MT and the relation between the

absorbency of bacteria at 600 nm and the concentration of bacteria are investigated in Figures. 1

and 2 available in supplementary file. Also, an equation is achieved to relate OD and number of

bacteria in the medium culture.

3.2. Effects of initial MT concentration at different time on MT removal

Time consumption is an important factor for each experimental work in batch or semi-batch

reactors. Figure 3 shows variation of MT concentration with time at specified pH, temperature

and bacteria OD. As it is clear from this figure, after approximately 300 minutes majority of MT

is removed. Another key parameter which influences the efficiency of system is initial

concentration of MT. Therefore experiments are performed to obtain variation of MT

concentration versus time at various initial MT concentration.

Figure 3.

By increasing the initial concentration of MT from 0.53 to 4.25 mM, the removal efficiency in

300 minute (at the lowest concentrations, the sulfur bacteria were substrate limited for a

significant part of the time, therefore efficiency is increased in shorter time ) is increased, but

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after 4.25 mM, by increasing the initial concentration of MT the removal efficiency is decreased,

as a result, more time is required for higher reduction.

3.3. Effect of dissolved oxygen on sulfur production

The only desired product in the biological MT removal is sulfur particle. In addition, the rate of

sulfate production decreases with reduction of DO and oxidation of MT to sulfur particles

increases consequently [45]. Thus, for more production of sulfur, the oxygen concentration

should be limited. The effect of DO values on sulfur production is illustrated in Figure 4. The

result show that, at initial MT concentration about 2.12 mM and DO value of 3.94 ppm only less

than 9% sulfur is produced, while at DO of 0.91 more than 70% of MT load is converted to

sulfur particles.

Figure 4.

3.4. Effect of time and initial concentration of MT on products

The effect of initial MT concentration on sulfur particle production is studied and the results are

shown in Figure 5(a). This fig shows that at low initial MT concentration the most part of MT is

converted to sulfate. Increasing the initial concentration of MT leads to production of more

sulfur particles and less sulfate [46].

The output products contain sulfate, elemental sulfur, thiosulfate, dimethyl disulfide and carbon

dioxide, however CO2 is one of the main products because of its existence in the surrendered gas

phase. Figure 5(b) indicates that at interval of 60-120 minutes, since initial concentration of MT

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is low, the amount of MT converted to sulfate and also elemental sulfur through biological

oxidation is low while, as it is clear from Figure 5(c), at high concentration of MT it doesn’t

observe. Figure 5(c) indicates that in first 120 minutes a bit biological oxidation is done, in fact

in this period of time bacteria adopted themselves by conditions to use available sulfur in

components as nutrients. Figure 5(b) reveals that at interval of 120-200 minutes, the amount of

produced sulfate is higher than elemental sulfur because at the first period of removal process

mediated reaction (Eq.5) improves fastly. But this conditions could be changed by consuming

time and increasing MT concentration as Figure 5(c). DMDS is one of materials which is

produced in first period of 120 minutes. Since more oxidation (non-biological) of DMDS needs

higher temperature and catalyst, after 120 minutes its production rate is decreased which shows

that at this condition, applied bacteria were able to reduce and break DMDS to sulfate and

elemental sulfur.

Figure 5.

4. Conclusion

In this study, biological removal of methanethiol (MT) in a semi-batch bioreactor is investigated.

The efficiency of the treatment process is checked at different operational conditions such as

temperature, pH, bacteria concentration (by Optical Density), dissolved oxygen, reaction time

and initial concentration of MT. Removal efficiency of MT increases by raising the bacteria

concentration to its optimum value for used its initial concentrations. The results show that MT

removal is decreased in pH higher than 8.2 and lower than 6 value. By increasing initial MT

concentration or at low dissolved oxygen, more parts of MT are converted to elemental sulfur,

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thus elemental sulfur selectivity increased and sulfate selectivity decreased. Under the optimal

conditions for the highest (8.51mM) and the lowest (0.53mM) concentration of MT, the

biological removal of MT after approximately 300 minutes reached about 89% and 94%

respectively, which shows that the applied method can be very effective for MT removal in

industrial applications.

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33. F.A.M. De Bok, R.C. Van Leerdam, B.P. Lomans, H. Smidt, P.N.L. Lens, A.J.H.

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42. F.P. Van den Ende and H. Van Gemerden, Sulfide oxidation under oxygen limitation by a

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Tables

Table 1. Examples of gas and refinery streams contaminated with MT, hydrogen sulfide and dimethyle disulfide.

Source Sulfur compound Concentration (mM) Reference

Anaerobic wastewater sludge digester MT 3.8-7.6e-3 [6]

H2S 1.8-3.6e-1

Landfill gas MT Up to 7.4e-2 [7, 8]

H2S Up to 4.4

Kraft paper production process MT 1.7e-1 [9, 10]

H2S 3.4e-2

DMDS 0.3e-2

Liquefied petroleum gas(LPG) MT 9 [11]

H2S 1.76

Refinery sulfidic spent caustics MT 0.01-0.42 [12]

H2S 0.91-1.97

Table 2. Medium culture of thiobacillus thioparus [35]

Main medium Vitamin mixture Trace metals

KH2PO4 2.0 g Thiamine-HCl.2H2O 10.0 mg Na2-EDTA 50.0 g

K2HPO4 2.0 g Nicotinic acid 20.0 mg ZnSO4·7H2O 11.0 g

NH4Cl 0.4 g Pyridoxine-HCl 20.0 mg CaCl2·2H2O 7.34 g

Na2CO3 0.4 g p-Aminobenzoic acid 10.0 mg MnCl2·4H2O 2.5 g

MgCl2·6H2O 0.2 g Riboflavin 20.0 mg CoCl2·6H2O 0.5 g

Vitamin solution 3.0 ml Ca-pantothenate 20.0 mg (NH4)6 Mo7O24.4H2O 0.5 g

Trace metals solution 1.0 ml Biotin 1.0 mg FeSO4·7H2O 5.0 g

Bromocresol purple sol 2.0 ml Vitamin B12 1.0 mg CuSO4·5H2O 0.2 g

Na2S2O3·5H2O 5.0 g Distilled water 1000 ml NaOH 11.0

Distilled water 1000 ml Distilled water 1000 ml

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Figure Captions

Figure 1. A schematic diagram of the experimental set-up. 1: nitrogen, 2: oxygen, 3 and 4: needle valve, 5:

bioreactor, 6: thermometer, 7: dissolved oxygen sensor, 8: pH sensor, 9: sampler, 10: ORP sensor, 11: acid tank, 12:

base tank, 13: air circulation compressor, 14: water bath.

Figure 2. Variation of uneliminated MT with temperature (a) and pH (b); Variation of

thiosulfate and sulfur production with pH (T=30˚C, bacteria OD= 0.5±0.04, initial conc. of MT=

2.12 mM, DO= 1ppm, time=300min)(c).

Figure 3. Variation of MT conc. with time at various initial MT conc.

Figure 4. Variation of S0 selectivity with Dissolved oxygen.

Figure 5. Variation of S0

production with initial MT concentration (a); Variation of

concentration with time for different components (b, c).

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Figures

Figure 1.

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Figure 2.

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Figure 3.

Figure 4.

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Figure 5.

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Supplementary file

Effects of bacteria concentration

Variation of uneliminated MT with initial MT concentration at various bacteria OD is shown in

Figure 1. At low MT concentrations, the bacterial oxidation process is slow due to substrate

limitation. At a constant initial MT concentration, higher OD of bacteria led to lower

uneliminated MT.

Figure 1. Variation of uneliminated MT with initial MT conc. at various bacteria OD.

In addition, a relation between the absorbency of bacteria at 600 nm and the concentration of

bacteria is needed. For this purpose, the method of colony forming unit (CFU) is used [44]. For

convenience the results are given as CFU/ml (colony-forming units per milliliter). The theory

behind the technique of CFU is to establish that a single bacterium can grow and become a

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colony, via binary fission. Then the colonies are counted and an equation is obtained to relate

OD and number of bacteria in the medium culture, as shown in Figure 2 and Eq. (1).

Figure 2. Relation between OD and colony forming units of medium with bacteria.

8

600 10)77.02.4(/ −=bacteria

ODmlCFU (1)

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