Biodegradation of Methyl Tertiary Butyl Ether (MTBE) By Tabinda Aftab

Introduction:

What is MTBE and what are its uses:

Methyl tertiary butyl ether (MTBE) is an organic compound with molecular formula (CH₃)₃COCH₃. It is flammable, volatile and colorless liquid that is sparingly soluble in water. It is used as an oxygenate and an additive in gasoline to increase its octane number. US has been using it since 1979 to substitute tetra ethyl lead to prevent engine knocking by increasing its octane rating. Octane number is a measure of performance of engine as by high octane number it can withstand more compression before igniting. Oxygenates help gasoline burn completely and increases oxidation while combustion. It has good blending characteristics and is low cost fuel, so it is preferred over other oxygenates by many refiners as it can also replace other gasoline components like aromatic benzene compounds and sulfur. (Anon 2009)

Why MTBE degradation is necessary:

It has minty odor that leads to unhealthy and unpleasant taste and odor in water. Water with low concentration of MTBE smells and tastes “nasty”, bitter or like Turpentine, for many people. Gasoline and heating oil travel through pipelines and are also distributed by truck to above ground and underground storage tanks. Underground storage tank leaks and spills provide major sources of MTBE. People also store gasoline in their cars, planes, boats, chain saws, vehicles and generators. Farm and residential releases and car accidents, spills and boats runoff also release and spread gasoline in the environment. MTBE moves through the soil and is dissolves in underground water easily. It takes longer to be broken down than other chemicals and remains persistent. As MTBE is much more soluble in water, its migration towards water reserves cannot be retarded. There have been some reports of acute symptoms, such as headaches, nausea, dizziness, and difficulty breathing, from people exposed during refueling to gasoline with higher levels of MTBE. There may be a threat of lifetime cancer in one in a million people having MTBE dissolved in their drinking water. (Owners 1995)

 

Biodegradation of MTBE:

Methyl tertiary butyl ether is relatively recalcitrant to oxidative or reductive microbial attack. This property is inherent in its chemical structure that is a combination of two bio-recalcitrant functional groups: ether link and the branched moiety. It yields low biomass and hardly sustains microbial growth. Instead of apparent recalcitrance in the environment, many laboratories have been reported its biodegradation by aerobic microbial consortium. It is well degraded over a wide pH range, a marked decrease in degradation has been observed below 4 and above 8 pH. (Administration, 2006)

Types of microbial degradation of MTBE:

MTBE can be biodegraded aerobically and an aerobically under controlled conditions. Recent studies have proved that under aerobic conditions specialized ad hoc microbial cultures mixtures are available like some pure cultures to degrade MTBE. It has been suggested that the difficulty in removing MTBE lies in the poor affinity between oxygen and MTBE degrading cultures. It is studied that difficulty in removing MTBE lies in the poor affinity between oxygen and MTBE degrading microbial cultures. For this anaerobic degradation is also done but only on trial base because of non efficiency. Much of the recent knowledge indicates that the process is highly oxygen dependent and effective for MTBE removal. So it can be classified as:

1. Biodegradation of MTBE under anaerobic conditions
2. Biodegradation of MTBE under aerobic conditions (Zanardini et al. 2002)

Biodegradation of MTBE under anaerobic conditions:

Anaerobic biodegradation is very controversial and only few researchers have reported degradation anaerobically involving extremely long incubation periods. Mormile et al. in 1994 investigated that Acetobacterium woodi and Eubacterium limosum are able to degrade phenyl methyl ethers under anaerobic conditions to find whether they can metabolize MTBE. Methyl butyl ether is completely degraded in both methanogenic and sulfate reducing conditions and only a small amount of methyl tertiary butyl ether underwent partial transformation to tertiary butanol after a long acclimatisation of 152 days. This formed product resists further degradation. Scientists reported this phenomenon as “rare occurrence”.

In the same years it was also reported that soils with low organic contents and low pH (5.5) supports anaerobic degradation of MTBE under methanogenic conditions. More recently, it has been reported that MTBE is attacked by microorganisms in iron reducing conditions, but this is not a well known phenomenon. (Sedran et al. 2002)

However recent laboratory scale investigations have confirmed the biodegradability of MTBE and tert-butyl alcohol (TBA) in iron-reducing conditions. These experiments were carried out using aquatic sediments in which three substances were added: 50mg/L MTBE, Fe (III) and humic substances. MTBE was depleted to below 1mg/L in 275 days. Sediments provided with humic substance and additional MTBE were able to degrade MTBE slowly but not completely; upto 60% only. Same sediments not amended with MTBE degraded TBA without any lag period. Humic substances play an important role in the degradation of organic matter. In fact, the Fe(III)-reducing microorganisms oxidise MTBE by transferring electrons to HS and, once reduced, the HS then abiotically transfers electrons to the Fe(III); HS is thus regenerated in an oxidized form. (Zanardini et al. 2002)

Mechanism of anaerobic MTBE biodegradation:

When MTBE biodegradation occurs under anaerobic conditions,   TBA frequently accumulates as a byproduct. As we currently do not have pure cultures   of anaerobic MTBE‐degrading microorganisms, the  biochemical reactions          involved in this process are not fully characterized. However, there is strong evidence that CO2‐utilizing anaerobic microorganisms known as acetogens play an important role in this process.

Acetogens are a diverse group of widely distributed bacteria that generate acetate (CH3COOH) as a product of their biodegradation activities. Based on what is known about the biodegradation of other ether‐containing compounds by acetogens, these organisms use the methoxy methyl group of MTBE as their electron donor and CO2 as their electron acceptor. The carbon from the methyl group is incorporated into acetate and both acetate and TBA are excreted as byproducts. (As et al. 2012).

2. Biodegradation of MTBE under aerobic conditions:

• Pure cultures:

Using pure cultures, MTBE, as the only carbon and energy source, is biodegraded in vitro. Three pure MTBE degrading bacterial cultures were isolated from activated sludge and the fruit of Ginko trees, the bacteria belonging to the genera Methylobacterium, Rhodococcus and Arthrobacter. Although these cultures were found to attack and transform 29% MTBE added to media at a concentration of 200 mg/L in two weeks, the mineralisation of the chemical was very low (8% after a week). Moreover, in the presence of more easily biodegradable carbon sources, the rate of MTBE degradation decreases significantly.

• PM-1:

A bacterial strain, (PM1) isolated is capable of utilizing MTBE, supplied at different concentrations, as the sole carbon and energy source. In presence of 20 mg/L the PM1 strain converts 46% of MTBE to CO2 in 5 days. It is the most widely studied bacteria for MTBE degradation than any other.

• Pseudomonas aeruginosa:

An MTBE degrading strain is identified as  Pseudomonas aeruginosa, from a consortium able to mineralise pentane. The strain metabolises MTBE only in the presence of pentane and there is no degradation of the alkyl ether without the alkane. Thus, the controlled addition of a metabolite to enhance MTBE degradation appears a most interesting possibility.

• Propane oxidising bacteria:

Steffan et al. (1997) tested the ability of several pure propane oxidising bacterial cultures to degrade gasoline oxygenates, including MTBE. After growth on propane, all the tested strains degraded MTBE, at the concentration of 20 mg/L. MTBE does not act as an effective growth substrate for propane oxidisers that prefer propane as carbon and energy source. MTBE is principally co-metabolically oxidised by mono-oxygenase enzyme activity induced by other substrates, such as the n–alkanes, under aerobic conditions. As oxygen availability increases, the MTBE degradation rate increases, following Michaelis-Menten kinetic. (Zhong et al. 2007)

• Mixed cultures:

MTBE biodegradation appears far more interesting using mixed cultures, rather than pure cultures. In fact with mixed cultures one microbial population is able to transform a compound into a metabolite that can then be taken over, and further degraded, by another population.

• BC-1 culture from bio-treating sludge:

An aerobic mixed bacterial culture, named BC-1 is isolated from a plant bio-treating sludge. This culture metabolises MTBE to CO2 (20%) and cell mass (40%). The BC-1 culture is able to sustain a population of autotrophic ammonia-oxidising bacteria that nitrify NH⁴⁺ and use the CO2 produced from the metabolism of MTBE as a carbon source. The BC-1 culture contained four or five microorganisms, including coryneiforms, pseudomonads and achromobacters. None of these isolates is able to grow when MTBE is the only carbon and energy source. However, the isolates grew in the presence of acetate and MTBE and degrade MTBE at a rate of 34 mg/g of cell per hour within 4 hours. The primary metabolite isolated from the MTBE breakdown is TBA. Other researchers have also reported that selected mixed cultures are able to degrade MTBE. (Sedran et al. 2002)

• Batch degradation:

Other mixed cultures degrade MTBE in batch at the concentration of 100 mg/L, the residual concentration at 80 hours ranging from below the detection limit to 50 μg/L. The specific activity of the consortium ranged from 7 to 52 mg MTBE/g dry weight per hour 79% of MTBE was converted in to carbon dioxide. In any case the growth of both mixed and pure cultures in the presence of MTBE as the only carbon and energy source is always poor, as evidenced by many authors. It is due to inefficient carbon assimilation pathway. (Kane et al. 2001)

Mechanism of biodegradation:

Propane-oxidising pure cultures have been demonstrated for their ability to degrade MTBE in aerobic conditions. Steps involved are:

• Production of tertiary butyl alcohol by oxidation of MTBE:

The first metabolite produced is tert-butyl alcohol (TBA). The conversion of MTBE into TBA seems to be mediated by a P-450 enzyme, most likely a mono-oxygenase, an enzyme that appears to have a broader host range. Experiments in the presence of P-450 cytochrome inhibitors revealed a significant decrease in MTBE conversion (64-85%). Further experiments performed in the absence of oxygen led to the same results. Besides propane-oxidising bacteria, Pseudomonas putida, also oxidises MTBE, as it has also P-450 enzyme.

• Oxidation of tertiary butyl alcohol:

The first metabolite, TBA, is further oxidised to 2-methyl-2-hydroxy-1-propanol and then to 2-hydroxyisobutyrate (HIBA), none of these are further degraded by propanotrophs. The accumulation of the two intermediates is a significant rate limiting step in the mineralisation of MTBE and TBA.

• Degradation of metabolites of TBA:

The further degradation of 2-methyl-2-hydroxy-1-propanol and hydroxyisobutyrate could require one of three proposed reactions: decarboxylation resulting in the formation of 2-propanol; dehydration resulting in the formation of methacrylic acid; or hydroxylation resulting in the formation of 2,3-dihydroxy-2-methyl propionic acid. (Fortin et al. 2001)

Evidences about the involvement of cytochrome P-450:

Involvement of the cytochrome P-450 in MTBE degradation is shown by the studies that mammalian P-450 can hydroxylate gaseous n-alkane and can o–dealkylate both diethylether. Hardison et al. (1997) studied the biodegradation of MTBE by a filamentous fungus, a Graphium sp. Strain. It does this under cometabolic conditions in the presence of pentane. They suggest that MTBE oxidation is initiated by reactions catalyzed by P450 cytochrome that lead to the breakage of the ether bond in this compound. Both tert-butyl formate (TBF) and TBA, MTBE degradation products, were detected, and the kinetics of the degradation pathway suggest that there is temporary TBF production preceding tert-butyl-alcohol accumulation; the TBF is converted to TBA, its further metabolism in this fungus still has not been discovered. (Steffan et al. 1997)

However, the results of MTBE degradation assays with Rhodococcus rhodochrous 116, that produced two P-450 mono-oxygenases, but did not degrade MTBE, indicate that not all P-450 enzymes can oxidise MTBE. Further studies regarding the MTBE degrading pathway, such as P-450 involvement, are needed to fully understand the exact role of the enzyme and its involvement. (Kane et al. 2001)

Factors affecting rate of biodegradation of MTBE:

1. Oxygen supply:

The effect of oxygen supply on MTBE degradation by PM1 in closed system has been investigated. It has been found that MTBE degradation is more rapid and complete when oxygen is continuously supplied through H₂O₂ in the closed system, and after 168 h, no MTBE can be detected. Meanwhile, only 40% MTBE is degraded after 168 h in the closed system without oxygen supply.

The oxygen supply also can accelerate the cell growth in the closed system. After 120 h, it is detected that the cell density increases about five times in the closed system with continuous oxygen supply by H₂O₂, while no significant change of cell density is detected without continuous oxygen supply. However, MTBE can be degraded by PM1 with very low rate after 120 h without H₂O₂.­­­­­­­­­ (Zhong et al. 2007)

1. Co-metabolite production:

The degradation effects of MTBE under various substrate conditions are different. The addition of diethyl ether, diisopropyl ether, ethanol and BTEX is found to have no effect on MTBE biodegradation in well-mixed reactors containing polyethylene porous pot for biomass retention. The presence of ethylbenzene or xylenes in the mixtures with MTBE completely inhibited MTBE degradation.

Liu et al. (2001) reported that the presence of alkanes or isoalkanes in polluted aquifers can enhance MTBE degradation. In this study, it was taken into account that the boiling points of methane, ethane and pentane are so low and these substances easily evaporate. It may affect experiment results and they would be hard to analyze, only hexane and cyclohexane were selected as co-metabolic substances to evaluate the effect of cometabolism on MTBE degradation. Study showed that the addition of hexane or cyclohexane inhibited MTBE degradation by PM1 in the closed system. It suggested that hexane and cyclohexane can be more easily utilized by PM1 than MTBE, and that no MTBE degradation is required for the metabolism and growth of PM1. (Zhong et al. 2007)

Biodegradation of MTBE by a novel strain, Mycobacterium austroafricanum

IFP 2012:

A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain is a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012. Ethyl tert-butyl ether is weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, are detected during growth on MTBE. A positive effect of Co^2_ during growth of M. austroafricanum IFP 2012 on HIBA is demonstrated. Degradation activity of this bacterium is induced by the addition of outer MTBE, and TBA was the good inducer.

Growth phases of Mycobacterium astroafricanum  on MTBE:

Growth of this bacterium alone on MTBE is slow and can be divided in two phases:

• First phase:

In the first phase, MTBE is degraded while TBA is accumulated, and this occurs at a nearly constant cell concentration.

• Second phase:

In the second phase, biomass is produced readily from the TBA that was accumulated during first phase. This is accompanied with the changes in CO₂ production over a time while the organism was growing on MTBE.

CO₂ is produced after a long lag phase from TBA. This lag phase corresponds to the conversion of MTBE to TBA. Another metabolite was also detected during the growth of Mycobacterium astroafricanum IFP 2012. This compound is identified as tertiary butyl formate (TBF). (François et al. 2012)

Involvement of monooxygenase:

Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by:

• the requirement for oxygen
• the production of propylene epoxide from propylene by MTBE- or TBAgrown cells, and
• the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene.

There is no cytochrome P450 in MTBE grown cells. Similar protein profiles are obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE grown cells.

Requirements of monooxygenase system in MTBE and TBA degradation:

No MTBE and TBA degradation activities could be observed in cell extracts from MTBE and

TBA-grown cells. The nature of the systems involved in the degradation of these compounds is investigated. Three important results of this study are as follows:

1. Necessity of oxygen:

First, oxygen is necessary for MTBE or TBA degradation by resting cells of M. austroafricanum IFP 2012. The average residual concentrations, under aerobic conditions, of MTBE and TBA after 24 h of incubation are 10% and 23%, respectively. While these are 45% and 75%, respectively, under anaerobic conditions.

2. Absence of P-450:

When crude extracts of MTBE-grown cells were used, no cytochrome P-450 was detected by observation of spectra obtained after reaction with carbon monoxide. The absence of cytochrome P-450 in cell extracts is consistent with production of propylene oxide from propylene by resting cells grown on MTBE, as alkenes are known inhibitors of cytochrome P-450.

3. Inhibition by acetylene:

Growth of M. austroafricanum IFP 2012 on MTBE and TBA was completely inhibited in the presence of acetylene at a concentration as low as 0.4% (vol/vol) in the gas phase. In contrast, growth on HIBA or LB was not affected whether acetylene was added or not. (François et al. 2012)

MTBE degradation by fluidized bed reactor (FBR):

Biomass retention is very effective in removing MTBE from the environment. Fluidized surface enriched with suitable microflora degrades MTBE efficiently. Fluidized bed reactors (FBRs) utilize granular activated carbon (GAC) as a biological attachment medium.

• They have excellent biomass retention because of extensive surface area and sheltering capabilities of GAC (granular activated carbon).
• GAC has the additional advantage of the capability to absorb shock loads of contaminants to the reactor. (Pruden et al. 2003)

Flavobacterium–Cytophaga and Beta Proteobacterium – PM1, are the two strains fund in the fluidized bed reactor (FBR) by denaturing gradient gel-electrophoresis (DGGE). The only MTBE degradation intermediate detected in the effluent is TBA, which is consistently measured to be below 1 μg/L. This is a promising finding as TBA regulations are now developing.

Membrane bioreactor (MBR):

Other biomass retaining reactors have been observed to achieve lower effluent concentrations, such as a membrane bioreactor (MBR) which achieves an average effluent MTBE concentration of less than 0.5 μg/L MTBE. An MBR is capable of retaining even virus particles and is much more effective than the FBR at biomass retention. This suggests that the biomass retaining properties of a GAC-FBR may not be sufficient for meeting strict effluent MTBE standards. The TBA concentrations, however, are significantly lower in the FBR than reported for the MBR, which may prove important if the health effects of TBA become a greater concern than the health effects of MTBE.

 

Effects of BTEX addition:

BTEX stands for benzene, toluene, ethyl benzene and xylene. MTBE contaminated water can be biologically treated using a fluidized bed reactor with or without BTEX addition. BTEX does not interfere with MTBE degradation because effluent concentration of MTBE before BTEX addition and after BTEX addition is comparable. Presence of PM1-like organisms before and after BTEX addition, along with stable removal of MTBE and BTEX suggests that these organisms are not inhibited by BTEX. This is an interesting observation considering that the seed culture that had no history of prior exposure to BTEX was capable of both MTBE and BTEX degradation. (Pruden et al. 2003)

Advantages of using native cultures during degradation:

Advantages of utilizing native bacteria include:

1. They are acclimated to the site groundwater chemistry, and are better adapted to the environmental stresses associated with the aquifer. They are more efficient at colonizing the surfaces of the GAC than inocula grown up in laboratory suspensions.
2. The native organisms are more capable of scavenging low levels of contaminants found in the influent to the bioreactor than the organisms that are grown up at the high substrate concentrations usually used to prepare inocula for bioaugmentation.
3. Using native organisms also eliminates costs associated with use of proprietary microbial inoculants in a remediation project.
4. Last but not least, re-injection (re-using) of valuable ground-water resources is much easier when indigenous bacteria are used for the biological treatment of the contaminant. (Hicks et al. 2014)

References:

2006. Administration, E.I., 2006. Eliminating MTBE in Gasoline in 2006. , pp.1–9.
2007. Anon, 2009. Methyl t-Butyl Ether ( MtBE ): Health Information Summary.
2008. As, O. et al., 2012. Tertiary Butyl Alcohol ( TBA ) Biodegradation Some Frequently Asked Questions.
2009. Fortin, N.Y. et al., 2001. Methyl tert-butyl ether (MTBE) degradation by a microbial consortium. Environmental microbiology, 3(6), pp.407–16. François, A. et al., 2012. Biodegradation of Methyl tert -Butyl Ether and Other Fuel Oxygenates by a New Strain , Mycobacterium austroafricanum IFP 2012. , 68(6), pp.2754–2762.
2010. Hicks, K.A. et al., 2014. Successful treatment of an MTBE-impacted aquifer using a bioreactor self-colonized by native aquifer bacteria, NIH Public Access. , 25(1), pp.41–53.
2011. Kane, S.R. et al., 2001. Aerobic Biodegradation of Methyl tert -Butyl Ether by Aquifer Bacteria from Leaking Underground Storage Tank Sites
2012. Owners, P.W., 1995. Drinking Water and MTBE : AGuide For Private Well Owners
2013. Pruden, a et al., 2003. Biodegradation of MTBE and BTEX in an aerobic fluidized bed reactor. Water science and technology : a journal of the International Association on Water Pollution Research, 47(9), pp.123–8.
2014. Sedran, M.A. et al., 2002. Effect of BTEX on Degradation of MTBE and TBA by Mixed Bacterial Consortium. , (September), pp.830–835.
2015. Steffan, R.J. et al., 1997. Biodegradation of the gasoline oxygenates methyl tert-butyl ether, ethyl tert-butyl ether, and
2016. tert-amyl methyl ether by propane-oxidizing bacteria. Applied and environmental microbiology, 63(11), pp.4216–22.
2017. Zanardini, E. et al., 2002. Methyl tert -butyl ether ( MTBE ) bioremediation studies. , 221, pp.207–221.
2018. Zhong, W. et al., 2007. Aerobic degradation of methyl tert-butyl ether by a Proteobacteria strain in a closed culture system. Journal of environmental sciences (China), 19(1), pp.18–22.

---

Author:

Tabinda Aftab

BS (Hon) Biotechnology

GCU Lahore, Pakistan