Acclimation of seed culture to degrade MTBE

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Acclimation of seed cultures to

degrade MTBE

Paul T. SunJuly 2007



Is MTBE biodegradable ? YES!

• Aerobic degradation of MTBE in nature is well known,

• Aerobic co-metabolism for MTBE degradation is alsoknown to occur with ethylene, phenol, even ammonia bio-

oxidation,• Anaerobic degradation or transformation of MTBE to TBA

has been known for a while but the mechanism is notunderstood.



• MTBE is a difficult compound to biodegrade due to itsmolecular structure with the tertiary methyl group connectingdirectly to an ether. The growth of the degrading culturesare known to be very slow, (doubling time around 2 to 5days)

• The yield is also very low, due to the required several stepsof substrate level monooxygenations making its bio-energetics very unfavorable for the bacteria to acquireenergy for growth.

• The micro-nutrient requirement of the culture is not very wellunderstood.

• The growth of MTBE degrading culture is known to be veryunstable in continuous culture studies. This can be due tothe predation by higher organisms in the reactor where theslow growing bacteria can not keep up with the rate themare consumed.

Aerobic degradation of MTBE



Acquiring seed culture from nature

sources

• The MTBE degraders in nature is rare. The presence of MTBEas a pollution source will have to be around for a long time

before MTBE degrading enzyme is induced.

• For example, in 2005, extensive soil samples were collected

from selected sites (mostly long term leaking gasoline retail

sites in California). The aerobic degradation activities weredetected only in 20% of the samples.

• Random samples from nature would have very low probability

of getting MTBE degradation activities.

• The most likely sources would be soil or groundwater samplesfrom MTBE contaminated sites, activated sludge from refinery

or chemical plants with MTBE-containing wastewater and

biofilter treating off gas containing MTBE. (see next graph)





Principle of cultivation

• Provide a good seed that has been exposed to a level of MTBE for a long period of time, as discussed previously,

• Provide a good environment for the degraders to grow,

i.e., DO, pH, nutrients, micronutrients, MTBE or TBA as

carbon source.• Prevent the wash out of the culture when conducting

soilds/liquid separation,

• Patience !!!!



The cultivation and maintaining a viable

MTBE degrading culture have three steps

1. Isolation and enrichment

2. Cultivating and maintaining a substantial seed culture,

3. Inoculation of the seed culture onto the GAC or other

media for real time applications.



Step 1 - Isolation and enrichment

techniques



Microcosm testing bottle or serum bottle

150 To 250 ml

syringe

Due to the volatile nature of MTBE and its slow degradation rate, typical

microbiological enrichment techniques, such as shake flasks, are not

applicable. Only gas tight microcosm or serum bottle technique is reliable.



Compound Concentration (g/L) Compound(trace elements) Concentration (mg/L)

K 2HPO4 4.27 CuCl2.2H2O 0.01

KH2PO4 3.47 CoCl2.2H2O 0.2

(NH4)2SO4 0.34 ZnSO4.7H2O 0.1

MgSO4.7H2O 0.46 MnCl2.4H2O 0.03

FeSO4 0.001 Na2MoO4.2H2O 0.03

CaCl2.2H2O 0.018 NiCl2.2H2O 0.02

Mineral Salt Solution

Sole carbon source: MTBE concentration should be in the range of 10

to 20 mg/l. Some studies has shown that with MTBE/TBA half and half

may be beneficial . Do not add any other major carbon source. (TBA isnot volatile so gas phase analysis won’t work.)

Adding 1 to 2 mg/l of yeast extract is to making sure that the need for organic

growth factors, such as vitamins, are met, whether the culture needs them or not

we are not sure, this is added for insurance purpose.

Composition of the enrichment solutions:



250ml20 g of seed soil or 20 ml of seed sludge

50 ml of mineral salt solutions with 10

mg/l of MTBE and 1 mg/l of yeast extract

Head space can be air or 50% air/O2 mixture

depends on the oxygen consumption of the

seeding material

Gas tight

Initialize the microcosm study – mixing the content

and making sure the bottle is gas tight



Shaking Table or magnetic stirrer

Periodically, take gas phase samples for the analysis

of MTBE. And using Henry’s constant to relate gas

phase MTBE to aqueous phase MTBE concentrations

(see next page)

Do not measure optical density or other biogrowth indicating concentrations. This is

due to the fact that the both growth and yield

would be low and these measurements are

not useful.

A non-biological bottle should be used as acontrol so that you are sure that the

experimental setup is not leaking.

The oxygen content should be

enough to keep the systemaerobic for the duration of the test.

This can be done by experience

or using micro-DO probe to

measure the content. (see next

pages)



Relationship between gas phase / liquid phase MTBE concentration

C, ppm v

S, mg/L

s, (mg/L)= C (ppmv) / 5.68

at 25C and 1 atm.

Shortcut to MTBE stripping calculation.lnk



The micro-DO or HBOD Probe

The HBOD Probe uses a fluorescence method to measure the concentration of oxygen in the

headspace of an HBOD test tube. The system is based on an optical fiber that illuminates a special thin-

film coating at the end of the probe with blue light. The coating fluoresces and is quenched in proportion

to the concentration of oxygen in the sample, and this signal is measured using separate optical cable.The device is a low-power system that is resistant to most interferences and is quite stable. Ordinarily,

the tubes must be kept in the dark during sampling (using a special container drilled to fit the tubes).

However, the probe can be made to work in ambient light. However, this increases the response time to

one to two minutes (versus only seconds in the dark).

The HBOD probe specifications:

Range: 0-100% oxygen in gas phase.

Response time: Uncoated tip: <1 sCoated: 60-90 seconds

Compensation: Temperature

Interferences: None (from CH4, acetone, moisture, or CO2)

Resolution: 0.1% at high O2

0.01% at low concentration

Calibration: 2-point calibration (air; oxygen-free air)

Stability: <0.05% per dayRe-calibration: Beginning of a new HBOD Probe test

Probe temperature range: -80° C to +110° C

Storage conditions: no specific requirements

Probe lifetime: 1 year (reconditioning available)

Prof Bruce Logan, Penn State University



control

Different seed sources

Plot the MTBE concentration chronologically



If some of the bottles show MTBE degradation activities, after the MTBE

concentration is reduced to low level, re-inject MTBE solution into the bottles

using syringe without opening the caps

Start the mixing again and monitor the gas

phase MTBE concentration to following the

degradation progress. (See next page)

One can continue these additions for 4 or 5

times. (DO may be limiting after a while)

Then the bottle solution needs to be

renewed. One has to stop the reaction and

open the bottle.



www.ehponline.org/members/2004/6939/6939.html

Adding fresh MTBE to the microcosm

http://www.ehponline.org/members/2004/6939/6939.html

http://www.ehponline.org/members/2004/6939/6939.html



1. The content of the bottle is poured out and to be

centrifuged to concentrate the solid material.

2. The supernatant solution is discarded or sent for

analysis

3. The concentrated pellet is re-suspended in freshmineral salt solutions and ready to be used as

seed for new microcosm tests.

4. Larger serum bottles can be used for cultivation,

5. These step by step enrichment techniques will

eventually increase the culture quantities to be

able to use in other laboratory experiments.

6. For larger scale experimental work, a more

sophisticated continuous culture for mass

production is needed.

How to transfer the cultures from one microcosm to the next?



Step 2 – Cultivating MTBE

degrading culture in an activated

sludge unit



Continuous Reactor Cultivation

• Run an activated sludge with very long sludge age, more than 30days

• Carefully design the aeration system so that a proper DOconcentration at 2 mg/L can be maintained without excessiveMTBE stripping.

• The feed should contain the proper amount of nutrients and withfeed MTBE concentration at 100 - 200 mg/L. Mineral salt innormal tap water was found to be sufficient for the cultivation. Noyeast extract was found to be necessary in our case.

• The COD/N/P ratio should be the usual 200/5/1. Nitrate can beused instead of ammonium as the nitrogen source. This willreduce oxygen consumption from nitrification.

• In order to prevent excessive predation by higher organisms in

this pristine environment, the system should be under anoxicconditions (no aeration but mixing), 2 hours, every other days or every week, depending on the condition.



•It is also recommended that another organic substrate

such as molasses at 100 mg/L of COD to be added as a

supplement to beef up the growth of the mixed liquor andalso share the burden of being consumed by the

predators.•The initial addition of an healthy activated sludge sample

from a refinery WWTP should be used to start with a

MLSS level at approximately 500 mg/L.•The system will start up without the MTBE inoculants for

several weeks for its to lineout and the results of

monitoring will determine the MTBE removal by air

stripping alone.•After the shake down of the system, the inoculants (from

Step 1) will be added into the aeration basin.•Long term monitoring starts.



Gas PhaseGC

N,P nutrient

solution

A typical Reactor setup

Blower

Offgas

Tap water

OrganicFeed solution, MTBEAnd Molasses

TotalFeed

6 ft

4’ 5 ½”

5’

4 SCFMW/ 5 air drops

Aeration Volume : 720 gallonsClarifier Volume: 189 gallonsClarifier surface area: 20 sq ftRated BOD loading: 1.25 lbs /day

AerationSection

clarifierSection

EffluentTank



P. T. Sun, J. P. Salanitro and W. T. Tang, “Fate and Biokinetics of Methly-t-Butyl

Ether in Activated Sludge Systems”, Presented at the 51st Purdue Industrial

Waste Conference, May 1996.





1. From the previous graph, one can see that for the activated sludge system to

maintain a healthy MTBE degradation system, the system has to have:

a) Long SRT more than 30 days or as long as possible.

b) High DO, 2 mg/L

c) Low stripping capability, use fine bubble diffuser system avoid surface

mechanical aerators. The system should also equipped with a slow

mixer to suspend the mixed liquor when aeration is periodically shut

down.

d) Feed concentration should be high, but preferably less than 400 mg/L. It

has been shown the at 300 mg/L there is a possibility of substrate

inhibition for some MTBE degraders.

2. Temperature should be at least 20 C to promote the fast growth of MTBE

degraders. They are like nitrifiers, it is difficult to get them started in winter.

But once they are established, they can sustain lower temperature.

3. The off gas MTBE concentration should be the best monitoring parameter.

Its concentration can be related to the development of MTBE biodegradation

easily.

4. The other test is to measure the biodegradation rate of the mixed liquor.

There are two different ways; the BOX test or the good old Microcosm test.



Cano, M.L., Wilcox,M.E.. 1995. Methods for the Determination of Biodegradation Rate

Constants for Volatile Organics in Activated Sludge Systems. Shell Development Company

Westhollow Technology Center Technical Progress Report WTC 109-95.

The BOX test to monitor MTBE

degradation activities of a mixed

liquor sample from an MTBE

treating activated sludge systemPlease see the reference for

details



60





•In harvesting the culture, one can withdraw ¾ of the totalmixed liquor without upsetting the system performance for

long.•One has to let the system to recuperate for two to threemonths before the next massive harvest to occur.

•However, if one only harvest half of the mixed liquor, then

one to two month recuperation period would be enough.



1000

Add Inocul

Removal by air stripping only



Step 3 - Inoculation onto BioGAC

(SGS US Proprietary information)

Documents

Acclimation of seed culture to degrade MTBE