Acclimation of seed cultures to

degrade MTBE

Paul T. Sun
July 2007
 Is MTBE biodegradable ? YES!

• Aerobic degradation of MTBE in nature is well known,

• Aerobic co-metabolism for MTBE degradation is also
known 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 not
understood.
 Aerobic degradation of MTBE

• MTBE is a difficult compound to biodegrade due to its

molecular structure with the tertiary methyl group connecting
directly to an ether. The growth of the degrading cultures
are known to be very slow, (doubling time around 2 to 5
days)
• The yield is also very low, due to the required several steps
of substrate level monooxygenations making its bio-
energetics very unfavorable for the bacteria to acquire
energy for growth.
• The micro-nutrient requirement of the culture is not very well
understood.
• The growth of MTBE degrading culture is known to be very
unstable in continuous culture studies. This can be due to
the predation by higher organisms in the reactor where the
slow growing bacteria can not keep up with the rate them
are consumed.
 Acquiring seed culture from nature
sources
• The MTBE degraders in nature is rare. The presence of MTBE
as 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 were
detected 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 samples
from MTBE contaminated sites, activated sludge from refinery
or chemical plants with MTBE-containing wastewater and
biofilter treating off gas containing MTBE. (see next graph)
The now famous PM1 culture was isolated from the LA County municipal
wastewater treatment plant. The system has a biofilter treating the off gas from the
activated sludge emissions. The off gas contains some level of MTBE for a while.
Notice the time scale, 400 days passed before the development of MTBE
degradation.
% removal

MTBE removal by Los Angeles County Sanitation Districts’ biofilter treating mixed liquor channel air.

LA County receives wastewater from 4 or 5 refineries in the Long Beach Area

 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
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.

Microcosm testing bottle or serum bottle

syringe

150 To 250 ml
 Composition of the enrichment solutions:
Mineral Salt Solution
Compound Concentration (g/L) Compound Concentration (mg/L)
(trace elements)

K2HPO4 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

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 is
not 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.
 Initialize the microcosm study – mixing the content
and making sure the bottle is gas tight
Gas tight

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

depends on the oxygen consumption of the
seeding material

50 ml of mineral salt solutions with 10

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

20 g of seed soil or 20 ml of seed sludge

250ml
 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 a

control so that you are sure that the
experimental setup is not leaking.

The oxygen content should be

enough to keep the system
aerobic for the duration of the test.
This can be done by experience
or using micro-DO probe to
measure the content. (see next
pages)
Shaking Table or magnetic stirrer
 Relationship between gas phase / liquid phase MTBE concentration

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

C, ppm v

at 25C and 1 atm.

S, mg/L

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 s
Coated: 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 day
Re-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

 Plot the MTBE concentration chronologically

control

Different seed sources

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

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 fresh
mineral 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.
 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 30
days
• Carefully design the aeration system so that a proper DO
concentration at 2 mg/L can be maintained without excessive
MTBE stripping.
• The feed should contain the proper amount of nutrients and with
feed MTBE concentration at 100 - 200 mg/L. Mineral salt in
normal tap water was found to be sufficient for the cultivation. No
yeast extract was found to be necessary in our case.
• The COD/N/P ratio should be the usual 200/5/1. Nitrate can be
used instead of ammonium as the nitrogen source. This will
reduce oxygen consumption from nitrification.
• In order to prevent excessive predation by higher organisms in
this pristine environment, the system should be under anoxic
conditions (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 and
also 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.
 A typical Reactor setup

N,P nutrient
solution
Offgas
Total Gas Phase
Feed GC

Tap water

5’
Organic
Feed solution, MTBE 4’ 5 ½”
And Molasses clarifier
Section

Blower Aeration
Section
4 SCFM
W/ 5 air drops
6 ft
Effluent
Aeration Volume : 720 gallons Tank
Clarifier Volume: 189 gallons
Clarifier surface area: 20 sq ft
Rated BOD loading: 1.25 lbs /day
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.
 The BOX test to monitor MTBE
degradation activities of a mixed
liquor sample from an MTBE
treating activated sludge system
Please see the reference for
details

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.
60
 Operating Methodology

• Do not waste sludge until the system MLSS concentration

is too high and effluent TSS gets too turbidity. Wasting to
harvest the bugs.
• When the system is in upset without apparent reasons and
when off gas and effluent concentrations get too high and
activities decrease. It is highly possible that the mixed
liquor get infected with very high number of predators,
protozoa, rotifers and worms.
• These higher organisms can be controlled by shutting
down the aeration system for period of 2 to 3 hours,
holding the MLSS in suspension by mixers. This should be
repeated until the predators number is under control.
•In harvesting the culture, one can withdraw ¾ of the total
mixed liquor without upsetting the system performance for
long.
•One has to let the system to recuperate for two to three
months 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.
Removal by air stripping only

Add Inocula
1000
Step 3 - Inoculation onto BioGAC
(SGS US Proprietary information)