8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 1/31
Acclimation of seed cultures to
degrade MTBE
Paul T. SunJuly 2007
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 2/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 3/31
• 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
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 4/31
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)
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 5/31
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 6/31
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 !!!!
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 7/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 8/31
Step 1 - Isolation and enrichment
techniques
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 9/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 10/31
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:
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 11/31
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
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 12/31
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)
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 13/31
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
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 14/31
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
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 15/31
control
Different seed sources
Plot the MTBE concentration chronologically
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 16/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 17/31
www.ehponline.org/members/2004/6939/6939.html
Adding fresh MTBE to the microcosm
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 18/31
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?
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 19/31
Step 2 – Cultivating MTBE
degrading culture in an activated
sludge unit
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 20/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 21/31
•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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 22/31
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
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 23/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 24/31
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 25/31
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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 26/31
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
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 27/31
60
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 28/31
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 29/31
•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.
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 30/31
1000
Add Inocul
Removal by air stripping only
8/7/2019 Acclimation of seed culture to degrade MTBE
http://slidepdf.com/reader/full/acclimation-of-seed-culture-to-degrade-mtbe 31/31
Step 3 - Inoculation onto BioGAC
(SGS US Proprietary information)