Kind: captions
Language: en
great okay all right um
good morning everybody um i'm gonna
today
continue with the bioremediation lecture
uh last week we talked about you know
biodegradation processes and
bioremediation
um today i'm going to talk about some
case studies
um i will rehash
review a little bit about basic
bioremediation and then we're going to
talk about
few case studies and then the last two
case studies i personally
involved in my research i'm going to
discuss that today
okay so just last week we defined
bioremediation is use of
biological organisms or their product to
enhance environmental cleanup
but specifically what we are most of the
time using is subsurface microorganisms
microorganism in the soil and below soil
to reduce the hazardous nature of
chemicals
convert them into harmless by-products
okay so you want to
make sure you degrade the chemical
ideally you want to mineralize the
chemical
convert them into in organic constituent
in some cases we biotransform because
it makes it less harmful than the parent
compound
so we also talked about natural
attenuation and enhanced
bioremediation by augmentation briefly
last week
so again the key point here you need to
have the bioremediation triangle
you need to make sure you bring all
these three elements together
pollutants organisms and the environment
whether it is soil water here um you
bring them all together that's the
engineering
challenge in real cleanup process
um so stages of biodegradation is you
know first of all you need to find out
whether there is microorganisms in
in the site that you want to clean up so
you take some sample
take with the lab isolate and uh look
for the
organisms identify
the micro microbial isolate and
working
technical difficulties yeah and then
after my optimization of degradation
condition
um you need to determine the efficiency
of your
microbial consortium and
and then you can identify the whole
metabolic pathway
then you want to make sure that you're
not activating um
instead of degrading the compound and if
you want to do some enzyme study you can
do enzyme study and then you can do
you know more detailed pathway that's
more basic aspect of it
just to know what is going on in the
site okay
so natural attenuation is not very
commonly used because not fast enough
is not complete enough um not frequently
occurring
um in in most of the site cases so
the current trend in cleanup process is
to stimulate
the organisms inside so most of the time
it's a biostimulation process
aiding um adding electron donor
are some limiting factors like nitrogen
and phosphorus
in some cases bioaugmentation is
necessary because the organism is not
there
okay so let's talk about uh one of the
uh
processes now coming up is
phytoremediation
and phytoremediation is again everybody
knows it is a
use of the plant for accumulation
or removal or conversion of pollutants
so phenomenation
broadly are using plants but there are a
lot of processes going on
you're stabilizing the chemical that is
called phytostabilization
you're water volatilizing the compound
from below ground
through a trans evaporation process
through the plant
it goes up in the atmosphere that is a
phyto volatilization
and then you have phytostimulation
you're providing the rhizospheric soil
microorganism interact with the chemical
and you stimulate
activity and then phytotransformation
plant enzyme itself can
transform some of the chemicals so that
is vital transformation
and then the last one is the one that
commonly used called phytoextraction
where the plant mobilizes the chemical
into the plant biomass
and then you harvest the plant and then
you incinerate
the plant to get another chemical so
there are so many processes going on in
fight remediation this next picture
you can see um
let me see you can see the plant uh
showing all this
phyto degradation right happening right
on the plant phyto volatilization
chemical go through the plant and then
you have stabilization
and extraction stimulation going on
so approximately there are 400 plant
species that have been classified as
hyper accumulators or heavy metals so
most of the time phytoelamination is
used to clean up
heavy metals um so it is it
it takes up the heavy metals and you can
harvest it and um
and sometimes you convert the heavy
metal from insoluble
soluble pump to insoluble form and you
can precipitate it so
so a lot of plants have been used some
of them are sunflower grasses
hemp alfalfa tobacco plant
then in aquatic system water hyacinth is
commonly used in
phytoremediation process
and again the root exudates of the
plants play an important role because
it stimulates microbial activity in
rhizospheric soil
and so it can activate some combination
of
phytoremediation and bioremediation
together in the
in the root zone of the plant okay and
then genetic engineering people are
using nowadays in
using greenhouse phytoremediation where
they put plant
and they put bacterial gene in plants
and then they grow the plants
to get rid of the chemicals so some of
them been very successful
but again it's very controlled condition
it's not very broadly used we're using
genetic engineering
exactly like in agriculture now we
produce bt
like bt carbon same um process
applied for cleanup
so biodimensional metal in the most of
the time we use phytoremediation
for metal removal heavy metal removal
okay
so we can immobilize the metal we can
bio
accumulate some metal using
polysaccharides and lipoproteins
we can precipitate the metal by
converting the metal
um from you know insoluble to soluble
form
and then we can solubilize the metal
using cedarophobes and organic acid and
various chemicals produced by plants
so contaminants that are aminable to
bioremediations
here's a list and readily degradable
compound are there the gasoline
component fuel oil we have ketone
alcohol aromatic compound then
and then two aromatic ring structure
compound like naphthalene
and some more degradable are creosote
and coal tars
and pentachlorophenol a lot of phenolic
compounds
and the compounds that are difficult to
degrade are chlorinated solvents like
tce carbon tetrachloride
some herbicides and pesticides a very
very recalcitrant compound that's going
to persist in the environment for a long
time our dioxins
and pcbs so i talked about the plants
uh microorganism have they produced the
pseudo-4 molecule to solubilize so here
is some of the example cedar falls
most of them are produced by pseudomonas
and pseudomonas tucciara
is commonly used in
you know solubilizing metal using cedar
force
and also the bacteria produce
biosurfactants
the biosurfactants are compound that has
both features hydrophobic and
hydrophilic which is non-polar and polar
region
in the same molecule that we call that
antipathic molecule
so biosurfactants are produced by
bacteria
cyanobacteria fungi and yeast and some
other
classes of biosurfactants are
glycolipids lipoprotein
phospholipid glycoprotein polymeric
biosurfactants
so these are widely used for compounds
that are not soluble
the compounds that are called dinapple
dense non
aqueous face liquid like tce um
to make it more soluble and bioavailable
for bacterial degradation
i have a problem today all right um
so the role of biosurfactan is again to
increase the bioavailability
of the compound that are hydrophobic and
also nutrient storage of molecules
um and save microbial cells from toxic
substances
it protects the microbes and an efflux
of harmful compound um the harmful
compound can be
you know excreted out quickly by the
bacteria so the bacteria will survive
in the presence of toxic nature of the
chemical
and also another features of by
surfactant is it's extracellular and
intracellular interaction
it helps in quantum sensing and in
biofilms of bacteria so those are some
of the
benefits of biosurfactant to the
microbes
so um so we take advantage of this
biosurfactant
because it's a chemical that are pseudo
solubilized by the
biosurfactant so it also reduces the
absorption of non-polar pollutant
to the surface of soil particles so
basically it increases the
bioavailability for degradation process
so again um quickly go through a couple
of this um
broad technique institute exo2 method
um into two is without
excavation of the site actually do we
dig up the site and
you know engineer the process i need to
make sure that you optimize the
condition
most of the time it's aeration sometimes
mostly oxygen limiting
and then you need to provide right ph um
moisture condition and nitrogen
phosphorus
limitation that's that's nutrient
addition
and sometimes you have to add
surfactants to make it bioavailable okay
so just again to to revisit from some
other process
um bioscience reactor can be used this
is really
fast um process so you can clean up
really quickly but it's going to cost
you a lot of money
um biopile is another process you can
put them in
big pile and create a optimum condition
for microbes
um and then we can use um land farming
uh commonly used landfall if the
compound is
leachable you have to put a liner
plastic liner and then you
create this landfarming condition for
cleanup
if the compound is not you know leeching
it's going to stay
um because of the soil condition you
don't have to dig up
and make a clay lay um prevent put up
put a leaching collecting system but you
don't have to do that
just you can go and do the land farming
without
disturbing too much um
we not only use bioremediation for in
soil and water but you can also use for
cleaning up the gaseous chemicals
using bioscrubber bio trickling filters
and slow sand or carbon filters so
if there is some gaseous form of
chemicals in subsurface condition
you can bring that up and put them in
scrubber and
and bacteria will use those volatile
compound as carbon so
so here's some of the example of
bioscrubber filters
is basically um using biological reactor
instead of putting influence
as a wastewater you're putting influence
as inlet gas
and that inlet gas is could be all those
volatile compound trapped
in your soil so you kind of collect them
and put them in this
bile scrubber or you can use low sand
carbon filters
and these are slush and carbon filters
you can have bacteria from a layer
biofilm layer when the chemical go
through
this layer and they basically use them
as carbon source
the layer contains heterogeneous
population of variety of organisms
including bacteria fungi rhodifier and
protozoa okay
so let's look at what kind of chemicals
mostly in soil and subsurface
contaminant
most of the time it's um oil related
compound like b
tax benzene tolerant ethyl benzene
xylene
we have thyrin and other
polynucleomatics
then we have chlorinated compound like
solvents like tce and carbon
tetrachloride
and then herbicide and pesticide and
then we have explosive chemical
also so here is a
picture to show the various sources
where they are coming from
i can come from um storage tank
underground storage tank can be leaking
from
a septic tank infiltration gallery
landfill leachate
and there are various sources that you
can get this pollution
industrial source and and landfills and
burial areas and dump sites okay
so the current issues i said with
gasoline use is
it's a widespread contamination this is
one of the major problem in us there are
more than one million
sites that need to be cleaned up and
it's a major threat to drinking water
um resource because it's going into
subsurface
and contaminate your drinking water
source um
like some other components of the fuels
are
carcinogenic you know if you cause
cancer and this problem is not
anymore but we still have pollution
before we added ethanol to gasoline we
in us we added this mtbe which is which
is called methyl tertial
detail ether which is which is the
oxygenated fuel oxygenator
to to burn the fuel better and have um
your tailpipe emission will be less
pollution
but now we have this problem because of
the gasoline with mtb
also in the ground water but currently
we come completely stopped mtv
we now use ethanol 10 ethanol in the
gasoline
so that's a typical fuel spill most of
the time from underground
um storage tank from gas stations all
right
so gas stations are old more than 50 to
100 years old
and they they're leaking into the ground
water and
this most of the lighter fragment of the
gasoline
float i call ellen apple light
non-aqueous face liquid
and then it forms this plume and it's
gonna spread in the groundwater
so that's a typical contaminated site
look like
um the second compound that is another
problem is chlorinated solvent these are
the uh the degreasing agent
um mostly it's tce trichloroethylene
this is just the opposite this is a d
napple because this compound is denser
than water
it will go to the bottom of the water
table and sink to the bottom
it has a low solubility so it will be a
permanent source of
pollution of groundwater and it's known
carcinogen
and it most of the time it's not a
primary carbon source for bacteria to
use it so here's a typical example of
the dna for tce
most of the time dry cleaners and also
the electronic factory manufacturing
factory because they use tce to
decrease the circuit board and also
aircraft manufacturing
and and also car factory wherever you
circuit boards are used they use tcu to
you know
decrease those parts um so those end up
in the ground water
and if you have this kind of problem you
have like clay lens
along with groundwater this tce collects
in the
clay lands and then it's going to drop
into the bottom of the water table
and become you know form a immiscible
layer
and then it slowly leaks into the
groundwater become a very permanent
source of pollution
uh forever and so this is one of the
major
um problem in the u.s for cleaning up tc
contamination
um so again this uh the challenge for
tce is
the the dean apple
is uh trapped on this clay lenses
and it's hard to get rid of and it
creates a soluble plume for years
okay that's a major problem with tce
contamination
so how do you clean up this kind of pun
contamination you do soil extraction you
do pump and treat
you can do physical or reactive barriers
to stop the
plume from spreading you can do air and
hydrogen sponging
and then you can use the microorganism
simulate the
production of good bacteria and then
of course we can give you a lot of money
you can use surfactant so surfactant
will make this chemical
more bioavailable so bio remediation is
the best way to do this tce
and oil cleanup in subsurface because of
these reasons there is no
additional disposal cause at low
maintenance
and does not create any eye shower and
capable of
impacting um large area
and decreasing site cleanup so so those
are some of the
um advantage of using bioremediation for
cleanup of tce
as well as oil spills in subsurface
condition
especially groundwater
um let's talk about um a little bit
about fundamentals of biodegradation one
more time
just there is no super bug we cannot use
them you cannot use genetically modified
organisms in cleanup
um all organisms our compounds
eventually will biodegrade but it takes
time
for bacteria to you know evolve enzyme
um it also needs specific conditions
such as the nutrient availability and
whatever is lacking inside
and again important part is the
contaminant should be bioavailable
if it is not bioavailable the bacteria
are not going to go
you know degrade this compound and then
you need to look for the limiting
factors mostly
oxygen nitrogen phosphorus those are
some other
uh limiting factor in in in the
in the contaminated site um
then we look for the what is happening
once you provide all that the organism
going to metabolize
and you need to look for the metabolic
activity of microbes
and and then you can choose between
aerobic and aerobic degradation if the
anaerobic works for you
just um you know simulate anaerobic
conditions and
enhance the microbes and then you can
reduce the
aqueous concentration of contaminant and
then you can reduce the contaminant mass
and these are some of the
advantage of using microorganism the
most significant process is
reduction of contaminant mass in the
system so
you permanently remove the chemicals so
you don't have a future liability
so the process is simple the organism
can using the contaminant and
mineralizing it and into carbon dioxide
water and other salt
and sometimes the biotransformation
happens
um example tce converted to dce okay
um so biodigression involves a lot of
extraction of energy from organic
molecules the bacteria get energy so
they oxidize the compound
and they grow on it and make more
biomass okay
so that's why this is very advantageous
using
microorganism for cleanup of these two
classes of chemicals
um again revisiting the degradation
process there are three ways to do it
the best way is microorganism to use the
contaminant as
primary carbon source okay if
that is the case then that's the easiest
way to go because the organism is using
the contaminants such as tca sole carbon
source
oils oil component and soil carbon
source
sometimes they use this component as
secondary substrate so they live on
other carbon source like glucose other
other easily available carbon source
um but they also use this as a secondary
substrate okay
um and also
the compound is not in high enough
concentration in the contaminated
site so they depend on other carbons and
then the third process is co-metabolic
condition so in this case you have to
provide
um additional carbon so such as glucose
to
enhance the bacterial biomass okay
so let's look at the requirements for
microbial growth you need to have
electron
accepted the electron donor could be the
contaminant itself
our carbon source could be contaminant
itself
got to have these conditions for
bacteria to be happy
right ph temperature and then the
limiting factor
and sometimes you have to put some trace
element this trace element most of the
time
subsurface is okay mostly the limiting
factor nitrogen phosphorus
and if it is aerobic the oxygen okay
so you gotta have various electron
acceptor options not only aerobic
you can use the uh like last class i
talked about nitrate reducing sulfur
reducing iron reducing condition if
there is no
oxygen available and then the
pollutant itself could be used as
electron donor
and the energy source the bacteria use
them
and convert them to co2 and water and
then they
use this to grow as well to make more
bacterial cell so again you can use both
aerobic and anaerobic
if oxygen is terminal accepted and the
process is aerobic
other processes are anaerobic depending
on what is
electron acceptor used in anaerobic
process
um in most cases bacteria can only use
one terminal electron acceptor
so if nitrate reducing condition if
nitrate is available it
is nitrate when nitrate is completely
current then
there's going to be a transition of
bacteria then go for next
electron acceptor condition available in
the site like sulfate or iron
so only one dominating
electron accepting condition most of the
time that's what
work in real world okay and then we have
faculty organism that could use
oxygen as well as other nitrogen
oxygen containing soil such as nitrate
and sulphate
um here is a bacterial metabolism
aerobically and anaerobically
you can use oxygen electron acceptor and
you can use co-metabolic condition using
additional carbon source
and anaerobically various salt you can
use
nitrate manganese ion sulfate and
methanogenic condition
carbon dioxide could be used so
this is what pictorially represented in
this um
slide so here is your dm the ellen apple
from your
underground storage gasoline tank when
it gets
get into this ground water um the first
organism that use up is aerobic organism
when all the oxygen is gone
then you can see the second group of
agonists and i take over his nitrate
reducing
condition then iron reducing conditions
sulfur reducing condition methanogenic
and this is the natural order of redox
potential
so this is how it works so when oxygen
run out
then you have a nitrate is used electron
acceptor then
iron then sulfate and carbon dioxide
which is the methanogenic conditions
so that's the order of redox condition
so here is from the literature you can
look at electron acceptor conditions
with different rank one to four one is
highly biodegradable four is not
biodegradable
so here's some other compound solvents
b-tags
pcbs and chlorinated compound like pce
and tce
you can see that aerobic condition most
of the time is highly
biodegradable but you can see under
anaerobic conditions
acetone can be degraded and this
tce and pce can be determined using
anaerobic conditions
and then b text also okay
um bio elimination practice basically
uh you need to make sure the physical
and chemical
and characteristics of the site you need
to understand
what kind of condition it is if it is
for example clay soil
then you're going to have a serious
problem because clay are going to
absorb a lot of this contaminant so it's
not going to be available
for by remediation you need to use some
free treatment to
make it available it will be more
expensive
and you understand the complete pathway
of what are the players
microbial players in the contaminated
site what kind of activity they do
and they again understand environmental
condition and once you know that then
you promote these
organism promote this condition and then
that's what this engineering aspect of
cleaning up is okay so this is just
basic
fundamental stuff once you know each
site what you need
then you go and provide that to the
particular site
again oxygen is the primary source most
of the contaminant sites are cleaned up
by aerobic process uh because of the
um the aerobic bacteria faster than
anaerobic
bacteria and here's a typical example
how much oxygen needed for
typical um you know benzene toluene
compound
and what product you get everything is
converted to carbon dioxide and
mineralized okay so in most of the cases
oxygen is all you need to supply and
everything else is in place okay
so how do you deliver oxygen um if you
don't have it in the site and you can
use hydrogen peroxide
pump the iron peroxide below ground and
you can
aerate your water and pump the aerated
water
instead of delivering oxygen in the
gaseous form
and you can do passively using membrane
and then you can also aerate
using in a pump to air it so those are
commonly used method
okay so in in a gaseous harmony
you usually when you use air you use
bio-venting process
i mentioned in the last class if your
contaminant is
above the water table between the
surface and
and the water table is in between there
above the water table
you use air sparging uh when the
contaminant is in the
below the water table move below the
water table so these are the some of the
common method used
for aerating the site okay
then um the dehalogenation reaction for
chlorinated solvent if your compound is
highly chlorinated like tce
um you're going to you know use
stripping the halogen
and put a hydrogen in its place
so here is a typical reaction of
halogenated compound
and the chlorine is removed reductively
and you put in chlorine in place you put
a hydrogen
so this is called d-halo respiration and
then anaerobic conditions and you can
also use co-metabolic conditions
so here what we are using is this um
tce itself has electron acceptor so
you're just replacing chlorine with the
electron
um so d-halo respiration in
chlorinated um contaminated site is very
commonly
used um as most of the time it ended up
tc converted into ethane which is
harmless product
and and we can enhance this by
co-metabolic process using
ec carbon you can have more bacteria
involved in this process
um high percentage electron donor goes
towards
dechlorination okay um
this dehydrogenation and depend on
hydrogen-producing bacteria to produce
hydrogen
because you need to have the electron
which is the preferred primary substrate
okay so this is a typical example here
is your microorganism
chlorinated solvent cells is the
electron acceptor you give electron
donor
and so it's going to consume and then
you replace this
chlorine with hydrogen so here's a pce
you can see one chlorine at a time is
replaced
and this is called reductive declination
process
until you get to ethane all the
chlorines are
replaced and then chlorine becomes
chloride and then it precipitate out it
become harmless
okay so and then um
sometimes the reaction stops depending
on you know
the condition for example pce can be
converted into tce and the reaction
stopped
tce is still you know a very
toxic uh equally toxic hpce
okay
so that's what i just talked about so if
the reaction stops from pc to tca
you're you're you know still having
toxic chemical in your contaminated site
and so this is more hazardous than the
parent compound
and so when same thing with t uh tcet
dc dc accumulates dc can be 50 times
more hazardous than tce
and sometimes you get vinyl chloride
from tce metabolism which is
carcinogenic so in anaerobic condition
this happens then you need to
promote aerobic bacteria to get rid of
this vinyl chloride so that's why you
need to understand the whole
process of what is going on in the site
so you cannot have just simply
monitoring parent compound
and say yeah i cleaned up side that's
not going to work you need to make sure
you show what happened to that compound
do the whole mass transfer and
complete mass balance what happened
and then the co-metabolic process it's a
photovoice transformation process uh
because the organism that using other
carbon cells the same enzyme could also
convert
the contaminant so so this is
commonly used and
but the problem is when you have this um
additional carbon source you're
promoting a lot of the other organisms
they're going to use
this carbon source but it's not going to
do anything to your contaminant so you
need to you have this
problem of so promoting the right
organisms but not promoting
all the organisms we call that wheat
bacterias like a weed
so you wanted to make sure that you
promote the organization that
carry out your particular d
chlorination or dehalogenation reaction
so here is again the example of
selective enhancement of reductive
dechlorination the competition for
hydrogen
is in subsurface is big because most of
the time the hydrogen can be used by
other organisms like methanogenic
bacteria can use
hydrogen to convert them to methane gas
or self-reducing bacteria
so you need to have this have hydrogen
partial pressure
and hydrogen balance and you want to
make sure this competition
is not going to um you know inhibit your
dechlorination process
so that's another um tight rope
uh you need to walk um when you're doing
this kind of cleanup
so what kind of electron donors you
could use um
if you are using co-metabolic condition
you can use a lot of alcohol and acids
um fatty acids so you can use
a lot of fermentable compounds you can
use like molasses
and and corn syrup and anything that is
cheaper you can use
um hydrogen is apparently your electron
donor
so hydrogen can become from a variety of
organic
substrates and so that's why you need to
do a small scale
field study a small scale studies before
you actually
do which donor is going to work for you
in the field electron donor
so here is a variety of donors commonly
used anywhere from acetate
to molasses to a lot of different
compound like
chi some other base from chicken menu
cheese whey corn stick liquor could be
used
and then you can use some fine chemicals
um you can use
depending on how expensive or how cheap
you want to do your cleanup process so
there's a lot of electron donors
commonly used
to do the cleanup um just to go through
the
enhanced bio attenuation process um
you can do the petroleum hydrocarbon
chlorinated um
solvents the electron accepted electron
donor category
so you can use aerobically anaerobically
depending on
your site and how you do this biospa
using oxygen slow release oxygen you can
use
aerobically and anaerobically can do
that
also and then chlorinated solvent
cleanup most of the time the electron
donors are as i said benzoic lactate and
molasses
molasses is very commonly used because
it's cheap
compared to other carbon sources okay
so those are i give you a general
um case studies for heavy metal for
phytoremediation
using pyro remediation and then tce
and oils will clean up below
surface so this is a lot of people are
done there's a lot of success stories
that i'm just giving a
general overview okay um
and some limitation in the process of
like competition
for electron um acceptor process so
now i'm going to talk about uh some
specific
cleanup that i was involved with and i
i i had some research project i
conducted in cleaning up
a particular explosive contaminated site
in louisiana
i'm going to share some of those results
with you and then i'm going to talk
about
the coastal oil spill that bp
oils cleanup and 2010 in louisiana which
i was involved
um also so i'm now going to talk about
this specific
cleanup case studies okay so this
particular one is
talking about cleaning up of explosive
contaminated style
in this particular ammunition site in
the town in
louisiana so this
tnt was considered as a recalcitrant
because this compound has been sitting
there since the second world war in from
1930s
during the production of second world
war this component started produced in
that site
but the compound is still there
at a high concentration nothing happened
to them so they
classified as recalcitrant um
and their recursion trend because of the
unusual substitution if you look at tnt
it has three nitro groups in its
structure
um of toluene and so toluene could be
easily degraded but when he added the
nitrous substituent
back to her hard time um you know
degrading that compound
so when you have this unusual
substitution like chlorine and other
halogen or nitro
and then then it become recursed
and and then other reasons also when you
have high molecular
size like plastics
um again why the vector didn't do the
cleaner because the
enzyme was not synthesized um
the compound uh the compound itself
it failed to enter the transport process
there is no suitable permeates to
transport them into the
cell the compound is unavailable
due to the limited limitation of
solubility tnt
maximum solubility is only 100 ppm in
water
and and then when you have high
concentration of the compound and
it's going to be toxic to the bacteria
so these are some of the
reason why this compound persisted for a
long time
so if you look at bacterial metabolism
just
a biochem 101. um
he has his metabolic pathway in bacteria
normally they use glucose and so when
they do that they
they do these glycolysis and then they
convert them to acetyl-coa and then the
restylane go to krebs cycle
and then the electron transport system
where when the electrons
are transferred from one electron period
to another electron carrier you bacteria
gets this atp
so from one glucose you can get 38 atp
molecule
and when you have lung chain fatty acid
what they do they do better oxidation
pathway
which is chopping two carbon at a time
converting them to acetyl-coa
and and take the rest of the crop cycle
so bacteria is not inventing any new
pathways
these are the pathways through the lung
evolutionary process
the organisms have evolved
into so when they see this new
chemical that we throw at them what they
do they
modify the structure so they make some
enzyme like
dioxygenase monooxygenase to
uh to break the ring and then once
they convert them to either an acid or
alcohol
then they shunt those metabolites into
these existing pathways so this is the
simple
basic mechanism of microbial physiology
so
they always use the existing pathways so
they never create any
new pathway for the new chemical we're
throwing at them
all bacteria does is modify the compound
and then try to fit them in any one of
these pathways
okay um in most of the time
the cleanup in real world is not pure
culture it is synergistic
degradation so there are two or three
groups of organisms
um they do one group organism do
biotransformation
and then convert the compound to some
metabolite
another group take them to next step
until it's completely mineralized so you
need to have
more than one group of organism doing
this reaction
until you eliminate it so very rarely
you see pure culture
in cleanup process most of the time it
is synergistic you need to have
a mixed culture of bacteria in cleanup
style
so this particular site i'm talking
about is contaminated with this three
explosive compound
tnt rdx and hmx so if you look at it
these are all have this nitro group
make it very unusual um substituent to
this molecule
that makes it hard and that makes it
persistent
in the environment for a long time okay
so there's a trinitrotoluvin this is
extra 135 tri nitro 135 triazine
commonly called royal demolition
explosive are rapid
detention explosive the two names that's
why it's called rdx
commonly known as plastic explosive then
we have hmx which is high melting
explosive
at the octo hydro one three five seven
tetra nitro one three five
seven tetra associate so these three
chemicals
persisted in this soil for more than 75
years never went away and this is this
particular site so i live
down here this particular site is up in
north
part of the state and the town is mindan
this particular set is more than you
know close to 16 000 acres
and it's been contaminated since 1941
and this is a big area um and
a lot of production said when it was
highly active there are eight
factories right there in the site that
produced this
various bombs chemical
and then they stopped production in 1994
and now currently it's because of this
contamination
and they fenced off this area and in a
caretaker status so the army
by law has to clean up the site before
they
um use this site for any other purpose
they can hand over to local government
but they cannot hand over until they
clean it up completely to the regulatory
standard
so so that's where this side is right
now um
so we got involved in doing some
research in this particular site
to look for how to clean up this site so
what we did
we did review the existing data we did a
very extensive physical chemical
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analysis of the site a collected sample
brought to the lab we characterized this
sample and we did small bio-treatability
study
and then we assessed and then we did the
bioremediation study
and then we recommended one method so
this
is what any fire immigration um
company they when they do the cleanup
this is what they do
they do look at what kind of data is
already there in the site and then you
collect sample and go through this
process and then finally execute the
cleanup
so our objective is to look for first
why these chemicals are there for such a
long time
are there microbes inside that can
clean up the microbes are not there okay
that was a very basic question
and so we if the microbes cannot use
this compound can we use this electron
donor i just talked about
provides a molasses to you know produce
more
bacteria maybe they will pick up these
compounds so
those are some basic simple questions we
ask
so we went to the site collected we
characterized we collected extensive
soil sample um and um
they did the analysis just to show hmax
and hplc
you know come at 3.3 uh minute retention
time
rdx come at 4.8 and tnt comes at 10.69
minutes
and then when you extract the soil and
you can see all three of them
so the hmx rdx and tnt it also has other
residues
and some of them are bomb chemicals some
of them are metabolized but
but we are concerned about these three
chemicals and how to clean up these
three chemicals in this particular site
so the this is a sixteenth almost
sixteen thousand acres so
um if you look at the t and d
contamination as four thousand to ten
thousand milligram per kilogram of soil
so the lowest range was four thousand
the highest was
at ten thousand and rdx is 800 to
1900 milligram per kilogram hmx is 600
to 900 milligram
this is the actual concentration of
these explosives in this soil
right the army wants to bring
this to below 200 and
the rdx to below 50 hmx to below
um 20. so that's their goal before they
can handle
so first thing we ask a simple
microbiology question we
put put the soil and enrich the soil
transferred isolated fuel colonies
and basic microbiology and then we use
the different conditions um
and these are some of the media we use
basic mineral cell media
and and then we have another condition
commodore body condition with glucose
in the lab and then the uh
once you isolated the you know stored
the bacteria and then did one by one
experiment okay
we isolated 22 pure cultures we screened
them all
each one of them it's a very tedious
time consuming process and i'm going to
just give share results with maybe two
bacteria
and it's no time to share all of them
so we staphylococcus is one of them and
bacillus
levolactic is another one
we grew this back pure culture in
different conditions so we had
obviously control no bacteria and then
we had used the
tnts soul carbon source no other carbon
in the
in the flask and if you look at tnt has
nitro groups so you could use tnt as a
nitrogen source or bacteria so we
eliminated the
ammonium salt from the media the only
source of nitrogen
is the tnt and then we used co-metabolic
condition where we had
glucose and the explosive together so
these are the various conditions we use
and then we analyze we monitor the
growth and we analyze the
compound in these cultures so here's so
quickly to go through
um if you look at staff various growth
conditions uh
control carbon source nitrogen shows
co-metabolic conditions
you can see the growth was better when
you have additional carbon source with
glucose and
explosives and then when you use them as
nitrogen source
they've actually used that tnt as
nitrogen source that somehow they
kind of remove the nitro group and use
the
um tnts nitrogen source
and then the carbon source also um
bacteria grow on it okay the best growth
obviously was co-metabolic conditions
okay
um and then the degradation of the t and
d
can see the same thing the chromatovoid
condition was faster
than carbon source conditions okay
same almost almost same um the trend for
baseless
various condition the core metabolic
work the best
komar walk removed tnt the best okay
but when you when you use the other
explosive
the story is different rdx cannot be
used as soul carbon source
and you use rdx as soon common source
nothing happened to the culture
only co-metabolic conditions showed
growth and co-metabolic conditions
removed
the rdx and it couldn't use other
nitrogen source um carbon source
both organisms did the same staph and
bacillus
so the take home is tnt if you
if you provide the right condition
vector could use some either
as carbon source or as nitrogen source
but of course we had a additional carbon
sources called metabolic it was faster
but the other two explosive the rdx and
then the hmx did the same thing
um hmx um only co-metabolic condition
worked
in both cases it is the same story for
10 of the isolates like other 10 um
isolate worked a little bit with hmx and
rdx
okay so that this most simple
enrichment study and isolated fuel
culture with each one of them
showed a general trend tnt if you put
the right condition can be
degraded without any trouble for the rdx
and hmx
though you need to provide additional
carbon source
otherwise this going to persist for a
long time which is what's happening
at the site okay so
this is a simple degradation study then
we looked at
how what kind of method will work
whether you need to use
land farming soil slurry reactor
composting
phytoremediation which method to
recommend so
we looked at the literature all the
different method we just
i just talked about um again to revisit
uh
bioremediation definition so we want to
intentionally
use the degradation process so we
enhance the microbial activity
and we again same
protocol for bioremediation go through
different
existing methods and then which method
to
use for treatability style
and so we choose these two method the
science layer reactor the reason for
that
is if the method is fast uh you can um
remember
we can remove the contaminant really
really fast but the disadvantage is that
is a capital intensive is going to cost
you more money
okay the uh land farming has a potential
for significant savings the operating
cost is less
but the reaction time is slow compared
to
so we want to compare these two and give
the army an option which one to use
okay so we set up a small
salsa reactor and we had a control there
was no molasses added
and then we had treatment we had
molasses added we had a slow
mixing and this was with 20 percent
slurry
you put soil and make it slurry with
water
all you add is just the molasses to the
treatment and normal ancestor
that's all we didn't do anything else
and then we just periodically took
sample
and analyze what's in the soil
so here's the average of those two
treatment and control so when he had
molasses added as co-metabolic
conditions
um you can see that at
tnd concentration going down really fast
in the red line and when there was no
molasses
it's going down but it's very very slow
right
so adding molasses point three percent
by volume um really helped your tnt went
down to the recommended
level uh within um six months of the
reactor operation so the army wants to
bring it
below 200 we did bring it to below 200
in six months um
surprisingly the rdx also went down
because it's uh
again it's because of we're enhancing
not
pure culture in the future it didn't
work but in the soil
adding molasses helped to bring in a lot
of microbial activity
so it took a long lag period nothing
happened to
the rdx for two months
after two months rdx in the soil went
down
in the in the reactor with molasses
and the reactor without molasses just
hang around nothing happened
right so co-metabolic condition the rdx
went down after two months of reactor
we were kind of first two months we kind
of whether to stop the experiment or
keep going
but we said let's go for it you know six
months like the tnt and
and eventually it went down okay and
um hmx is also
uh took a very long time before you can
see any uh decrease in
concentration um so with without
molasses it just
persisted and uh vic molasses that
took almost three months before you can
see big drop in
hmis so so the this small simple study
um tell us that by putting molasses
and mixing you can remove these three
chemicals in a reactor uh within
six months
okay that's the that's the result from
this study and then you want to do the
landforming we started out in the small
scale
so just to simulate the land farming
process in this
pan we put the contaminated soil and
in this pan we had molasses we just
fled with molasses and then we drained
the molasses and then we
we till the soil by aerating the soil
in the control normal i said just the
water right
um point three percent molasses in these
two pan
no no malasa just the water here okay
uh again the same kind of result um when
you
had a molasses treated land farming pans
you can see this tnt go down but it was
not fast it was not as low as
precisely reactor satellite given down
below 200
but it did it did go down in the
landform
right because you can tell the
landfarming is a solid
face system whereas in slurry reactor
we make it a slurry mixing so you have
better mass transfer
so the reaction was faster there here
reaction was slow
so but you can see this eventually went
down
um significantly okay
um same story with rdx um it um
it took a long time in the land farming
pants it it started to go down okay
after
four months in landform
and hmx also it took a very long time
for hmx to go down but hmx eventually
also started to go down so what does
tell us
if you if you have a lot of time and you
don't want to have
somebody holding gun on you to clean
this site right away
you can use land farming this will take
more
maybe two three years to clean it up but
it eventually
will clean up these chemicals so that's
basically what but you need to apply
these molasses
every month once a month in the field
so the summary of these two uh
bioremediation
science very landforming you can see
this removal of this chemical
99 percent in tnt and slurry versus 82
rdx 97 investor 90 hms 87.71 so
both of them removed slurry reactor
remote um
better and faster than landfall
okay so now all i showed was
um just the removal of the compound i
had to
do metabolic pathway and make sure there
is
no activation going on make sure all
these compounds are
converted to harmless product right so
we did the mass balance study by
spiking radio label tnt and then
monitor the radioactivity of c14 tnt
and you can see in the control most of
the tnt
is still there and you see some
metabolite these are the
biotransformation product of four amino
di nitro toluene two amino di nitro
toluene they are produced in the control
but when you have molasses you can see
this
um 22 percent of the tnt is mineralized
to carbon dioxide
which is spectacular by 22 percent
mineralization
and 23 percent of the already labeled
tnt
ended up in making more biomass
bacteria use them to make more bacteria
in in you know in the cell and then we
have
a lot of other metabolite accumulating
and but these two account for almost
fifty percent
of co2 as well as um
accumulating in biomass almost the same
story for land farming
most of them without molasses remind
in the soil nothing happened to the
compound but when you add molasses you
produce again
mco2 and produce the the tca which is
the biomass
preservative material okay so this this
tells us that tnt is mineralized
and then this is a construction of
bacteria not through culture
the puke culture only i showed you a few
cultures but this one has a lot of
bacteria in the soil every
all the organisms are enriched and they
are
doing a fantastic job of mineralizing
this
explosive so this is the kind of aerobic
process because you're mixing your
oxygen going in there and then you're
also
land farming you turn the soil over so
you're actually um increasing the
aerobic factory we want to know what is
happening in anaerobic condition
because we take one single soil
particles you can find
oxygen gradation the surface of the
particle is aerobic as you go
into the middle part of this anaerobic
so if you
can promote anaerobic activity you want
to know what kind
of dnd degradation happens in the soil
so we promoted sulfur reducing bacteria
by providing 20 millimoles sulfate
and and then we developed a consortium
of disulfurious species there are four
different bacteria
and they can certainly use tnt as soul
carbon source they
so there are anaerobic bacteria in the
soil
so you know so if you provide some
sulfate
and the bacteria will pick up this tnt
soul carbon source so here is uh
those consortium we use in the lab um
this one is
with the metabolic condition we use
pyruvate
substrate go substrate and then this one
is without pyruvate just the tnt
um and this is just a control without
bacteria
so you can see the growth and you can
see
the tnt go down so metabolic of course
went
fast and sold carbon source you know
like four weeks uh almost before it went
to zero in the culture
so this tells us that if you as i said
at the beginning
you can provide some electron acceptor
so like nitrate sulfate
so oxygen of course is good but
there are some anaerobic pockets so if
you want to encourage that bacteria
if there is some sulfate already there
why not
you enhance the bacteria so that's what
we did here
and then we created a solid reactor
anaerobic slurry reactor
and enhance the activity and monitor tnt
and you can see in the solar reactor
the 6 000 milligram per kilogram of tnt
went down within 100 days in the
anaerobic conditions
and the control when there is no
bacteria
and nothing happened to this dnt so
there's no
abiotic activity mostly biotic activity
that
taking over an anaerobic process again
we did the ready level study we spiked
some radial level tnt and accounted for
complete mass balance um
this particular anaerobic bacteria is a
what we call is incomplete oxidizer it
doesn't take the
organic compound all the way to co2 so
what it did was it
um if you look at this column
um the real reading label tnt 27
of it converted to the cell biomass
most of them are converted to acetic
acid so
the acetic acid accumulated in the
system 47.5
of tnt is converted acetic acid
this particular consortium doesn't have
that the final step
to oxidize acetic acid to carbon dioxide
so that's our
end product from tnt to acetic acid
then we produce nitrogen this has
another metabolite nitrobenzoic acid
butyric acid you know pentanoic acid and
these are biotransformation product
and so based on this we constructed the
metabolic pathway so this is what's
going on anaerobically
so here is your tnt the the thing
happening here
is this transformation reduction of the
nitro grouped
amino group so this becomes four amino
to six dinitro
and then in the second position nitro
group is reduced this will become two
amino
and then two and four amino already
become two for diamino
and then we couldn't catch some other
metabolite the next metabolic
carbohydrate is nitrobenzoic acid so we
are
we are um uh kind of suspecting this
amino group is clean there is a
deamination reaction
this amino could be used as a nitrogen
source for bacteria
and then the methyl group is converted
to carboxylic acid and we we found
nitrobenzoic acid
and then after that the ring cleavage
and once the ring is cleaned and you
have
cyclohexanone and then fatty acids once
you have fatty acid then bad oxidation
pathway
to carbon at a time chopped and
converted acetyl-coa
that goes to krupp cycle and then all
the way
it's everything is converted to acetic
acid so
you know pentanoid to betray propionic
and
acetic acid so this is of the
end product from harmful tnt
to harmless acetic acid acetic acid
could be used by aerobic bacteria as a
carbon source
right so this is what's happening
anaerobically in that soil
so that's particular um
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site we recommended two choices we gave
the
army you could use a slight slurry
reactor if you want to do a fast cleanup
if you have a lot of time to clean up
you use landform method there's a
trade-off between
the time versus the cost of cleanup
and what they did was they they divided
the site into several areas the
immediate um
area that if they want to hand over to
the um
to the local government they they did
the reactor
and then some area that uh protected
for nature conservancy
project they went with the uh land
farming method
so that's that's what that site has been
cleaned up
okay um i'm now can i continue with the
one more case study
in chandra
you have nine yes yes
time yes we are still trying okay so
this is
i was involved for a real cleanup and we
did the lab study we did the
treatability study and we recommended
the method the method has been executed
and out of the 15 000 acres i
i was last told um half of it cleaned up
seven thousand acres being cleaned up
and handed over to the local go
okay so now let's talk about the oil
spill
right this um some of you heard about
this that
10 years ago little over 10 years ago on
april 10
2010 we had a big oil spill right in our
backyard
i live 75 miles from this
actual actual site where this
bp oil blew up and so this was in the
gulf of mexico
and uh some of the history behind what
happened is this is what happened
and the explosion happened in deepwater
rice and drilling platform
11 people died and it took
until it happened in april until
september 17th to stop the oil
to cap the oil and it took a long time
to
contain and during that time we had
fifty thousand to sixty thousand barrels
of crude oil
uh flow uh happen
uh every day per day okay it
covered white area 2500 square miles
and by the time they capped the well
there are
170 million gallons of oil spilled in
the gulf of mexico
this is the largest marine spill in us
history okay 10 years ago um
most of more than 50 of them were
dispersed at the depth
like when 10 000 feet below 5000 feet to
the
surface and then below okay so during
that time they used 1.8 million gallons
or this person which is commercial one
called corexit and 6500 that was the
dispersion they used from nalco
chemicals and they used the dispersion
at the site
where the oil is coming at the
below the seabed under the seabed
and also used aerially they applied an
open ocean
so as a result a lot of things happen
people are affected
economically because all the fisheries
shut down
and like a lot of people lost their
livelihood
environment was severely damaged the
whole state
suffered economically and then we had
problem
geological problems in the gulf of
mexico because of this oil
problem um some of them are
chronic problems some of them are acute
so most of most of the acute problem
um is gone but this chronic problem
still persists people still have this
effect in here
okay um and just i just want to make a
point that
in gulf of mexico we have a natural
seepage of oil
in the seabed every year we have 50
million gallons
of oil naturally comes out okay and
that's a part of the natural process
okay
but this all of this oil came all at a
certain
time all at once that what's the problem
so when it's slowly leaking
microorganism takes care of that okay
so first thing what they did was they
want to
kind of on on-site what institute
burning a wire so they whatever oil that
accumulated on this
ocean surface they burn it up so
they light up and they use some
accelerant to burn the oil so you can
see this big
plume of burning of this crude oil
and then they contain it they want to
contain it they use these big
uh you know skim skimmers and containers
to contain this oil
getting into the shore okay they use
massive operation people spend billions
and billions of dollars
and so this is how the site looked like
um pictorially
schematically so this is where they
drove um
this is the sea surface 5000 feet to the
um the sea floor
and then they went 13 000 feet drill to
reach this oil
all right so that's where this um oil
makanda so in the makanda reservoir
the problem was it has high methane
content
and there was some problem with this
automatic
shedding valve that they were not
properly taken care of
so this methane built up and the methane
blew up
and as a result we had a big explosion
and then the whole thing uh there's no
containment
so that's what happened okay so a lot of
methane in this oil natural gas
so is there some picture to show you the
damage you can see
oil on the surface on the
shoreline most of the shore are wetland
salt marshes saltmasters are covered and
this is the gulf of mexico
it's a florida um louisiana right here
and this happened here so the current
took the oil everywhere it went all the
way to
uk atlantic uh
the loop current took them all the way
to uk
see the damage a lot of wildlife got
killed
and a lot of oil on the coastal marsh
area and then you can see the uh
activity on the uh surface of the ocean
okay
and it's the 707 721 miles of coastline
of louisiana was affected a lot of them
are this
marsh wetland and maybe a lot of
wetlands highly productive at land
a very good fishery spots so all the
fisheries sat down
they cannot fish this area and just to
show you more pictures what they do they
they constructed some sand berms to
to to catch the oil so it won't reach
these fishery spots
and then they put this um also the
booms to contain oil and this is what's
happening and uh
you can see the damage when they do this
kind of activity to contain oil
okay um so
more pictures to show you the they have
small small barrier islands
they cover the whole barrier islands
with this um boom
to make sure it won't reach because
these are a bird sanctuary
uh migratory bird land here they want to
protect this
and and they applied this dispersing
agent right
open open ocean they're blind okay
so just if you look at the date there
are different days
there's a june picture and it's a july
picture
it looks like no oil right
but when you go close and take sample
the oil is still at the bottom
or you can see within a month most of
the
volatiles were evaporated and uh
some of the wave action took the oil
back into the ocean but
there is i'm going to show some close
picture but still some oil
here after the july picture
um so you can see a lot of spa a lot of
those local ecosystem
this is oyster this is where they
harvest oysters
and they're all completely covered with
oil actually they can't use them at all
um for a long time we couldn't harvest
oysters
so you can see this the area that i
showed you from
long shot you don't see it but when you
actually go close
you can see the oil still there right in
september it was still there you can see
the
initial oil and then gradually you can
see the mark everywhere
more pictures just to show you the
damage to the ecosystem
a lot of oil in the shore and let your
wet
marsh over there
this is how it looks like in close up
and this is the dispersant without this
person when you don't have this person
after you apply dispersion the oil is
dispersed okay
of course this person has its own toxic
effect on the ecosystem but
um at least it broke the
oil into smaller particles and dispersed
okay
just to show you what happened in a
picture um
so when you have oil like this when you
add this person
it's it actually dispersed i will be it
will be clear
within few hours few minutes the
dispersion has a really
very good effect on oil
so what happened the mass balance of oil
according to nova
um all the uh 170 million
gallon of oil that spilled and
17 dp recovered right at the wellhead
they sent some big pipeline to catch the
oil
and the five percent they did the
open-air burning
and then they a lot of small boats went
and skimmed the oil using some pump
[Music]
sea water and oil mixed together but
then later they separated
then chemical dispersion using this
corrects it and it dispersed into open
ocean and went into this loop current
into
atlantic ocean um 16 percent is
naturally dispersed
um because of wave action and
85 percent is either evaporated
are dissolved in in the ocean but we
still have this 25
oil in the coastal bottom
so every time there's a big hurricane or
big
wave action this oil can you know come
back
so this this is what they're worried
about this 25 percent gonna have this
um what do you call that chronic effect
on the ecosystem so
this is where we come in uh so
how are you going to get rid of this 25
percent that is buried in this
coastal ecosystem okay
so my our research project was to just
to help the oyster farmers because the
oyster industry
in the southeast louisiana is big and
it's a small form
uh not a big operation these are all
small
individual oyster farmers so they
is a good industry in new orleans is
famous for oyster
um seafood and so we uh
we uh went into these two sites um
um where the oyster is um we collected
the sample and brought to the lab and
did our basic studies
okay so this is how it looks like when
we went to
take example it looks pristine it looks
clean
from from uh uh distant okay when you
when you go and collect samples
at the bottom because it's all at the
bottom of the
coastal area you can find oil and this
is oyster sample
and then we collect the sediment oyster
and water
and we did the analysis of oil and
and then we did the enrichment study for
bacterial activity and how the oil can
be cleaned up
so what we did was we did the redox
conditions of the sediment
the sediment is not aerobic but sediment
has a lot of
nitrate and sulphur reducing bacteria if
you
just take one inch of the sediment you
can smell the hydrogen sulfide
so there's a lot of sulfuridation
bacteria there
so if you put enough sulfate is electron
acceptor maybe this bacteria will take
care of the oil
right so we we set up various conditions
nitro reducing sulfate reducing iron
reducing methanogenic fermenting and
then we put all of them together we call
it mixed electron acceptor we want to
promote all the bacterial activity
in one condition and see whether that
will do anything good
okay uh we set up a satellite reactor
and then we did the soil
column study we took a core sample and
then we
mimic each of these conditions in core
sample
um so these are the um the slurry
reactor conditions
and we had a duplicate culture bottles
and because of two sides there's so many
bottles so we couldn't do a triplicate
um and then every sampling event we
sacrificed
each um duplicate bottle and we did the
total petrol hydrocarbon so
we set up the condition and then we took
the whole
bottle for every week and then we did
the analysis
we followed the epa protocol the
experimental setup showing different
culture conditions in big shaker
quickly the result we monitored the
total
petroleum hydrocarbon we didn't we
didn't care about analyzing
how much is aromatic how much is um
you know aliphatic and polyaromatic we
just did the one
total carbon all the total area in the
gc was taken
and you can see when you have um the
mixed condition
when you have all the electron acceptor
the degradation was
better it was surprising i thought at
the beginning of the lecture
i told you right generally
microorganisms
wanted electronics at a time this is
totally opposite
i said oxygen first nitro reduces come
then
you know ion reducing bacteria but in
this case when we put all of them
electro accepted that condition removed
oil
better and and then you can see this
blue line the blue line is the
fermenting condition that means
no electron acceptor it's just anaerobic
we want to make sure the oil itself
could be used as
electron acceptor then so it can
you know degrade and put the electron in
the oil
metabolites okay and this of course
control
um no electron acceptor at all
um and then when you put individual
electronics after the sulfate reducing
condition so out of the mixed electronic
set condition that it looks like
the sulfate reducing bacteria is doing
the bulk of the job so when you have
only the sulfate
you can see 55 oil removal
and when you have nitrate um
you have 30 oil removal so if we combine
these two
it accounts for the mixed electronic
except conditioner 80 percent oil
removal so
this looks like there is a some kind of
synergistic activity between these two
groups of bacteria in this sediment
and then based on this quick study of
four months
then we set up this core soil column
experiment where we
put several soil columns and mimic the
same
conditions and this soil come from the
site i showed you we dig up the core
sample and put it in there
all we did was we pumped sulphate
20 millimole in sulfur reducing
condition nitro reducing condition we
put
nitrate and mixed condition put them all
together okay
so quick result um we can see the
the co2 production uh in the
i'm only showing the mixed mixed
condition you can see the
co2 production going up um um
really well um we dose it we dose it and
based on the wave action so when when
there is the high tide low tide
and wave action we drain the column
so you can see no co2 when we drain it
and then when we flood it this
water comes up so microorganisms are
working on this oil and they
and and and converting them to carbon
axon
and then we also monitored methane so
you can see the methane
compared to control and mixed
electronics of condition produce more
methane
okay and different periods right
and then we did the count how many
bacteria are there
um using this uh some kind of uh
gross um analysis of
making the media to promote only this
kind of group of bacteria so it's not
very accurate but it just gives a bulk
part
figure of what is going on in this
condition so when you have all the
electron acceptor
that's where you have a lot of bacteria
growing and
in the soil column uh when you have this
um
um the blue line which is the sulfur
reducing
is the second best and then you have the
the nitro the green line the
methanogenic condition
and then you have the nitro reducing
condition okay
so this is the order so when you have
all the electronics that are triggered
that you have
better bacterial growth in the soil
column
and then we have sulfate methanogenic
and
nitro reducing conditions and just to
show you what happened in the soil
column at the beginning you can see the
lot of this crude oil
fraction in the column and then after
160 days you can see the same soil
column
uh mixed electronics or condition you
can see the signature peaks coming down
um after 290 days you can see almost
minuscule amount of carbon left there
and then this is the removal activity in
the soil column
and you can see mixed electron acceptor
worked
better than the slurry reactor in the
soil column work better than slurry but
the operation was
almost you know 50 days compared to
in the slow reactor result was 120 days
so um the carbon removal was really good
in
mixed electron acceptor condition and
then he went down
sulphate and nitrate reducing conditions
and just took the best condition we put
this
bacterial growth with the percent
removal the mixed electron acceptor
condition you see the
the mimicking bacterial growth with the
highest removal activities when they are
actively growing
and when the dividing cells in the soil
column
you see the best removal activity so and
then it reached
the point where most of the carbon is
gone and you can see the bacteria
started to go down the number of
bacteria are going to go down like
two order of magnitude down going down
so
so this just tell us that um
if you provide you don't have to provide
all the electronics you provide
at least sulfate and nitrate there are
already there some of it
but according to our study need of 20
millimoles
so we need to kind of formulate this to
add this to this site uh maybe this will
speed up the
actual cleanup of those 25 percent oil
in the coastal
coastal sediments okay so it just took
some electron microscope you see a lot
of bacterial
activity in the soil column all kinds of
different
heterogeneous populations
so this study basically showed us that
um
again at the beginning i said the
electron donor electron acceptor
you need to know which one to add and if
you find the right mix
and the bacteria will do the job that's
what we did here
so the nitrate and sulfate reducing
condition worked
better for us when you add both of them
mixed condition work really really
better so
um there was a little bit of surprise to
us i thought one condition worked better
than
other but mixed condition worked really
well also
um so the funding for this work both of
the study come from department of
defense and army corps of engineers and
national science foundation and lucian
board a region
so so that's the two
um case studies i was personally
involved
i'm just sharing the data with you so
i will stop here and i'm glad to take
any questions
okay thank you raj uh
beautifully there there's some
there are some uh questions right
probably okay
like is you stop you want me to
stop sharing this one yes okay yes
okay okay good
it's bad wait i mean
just a minute okay
all right
so i want to go through the first
question okay
so yeah it's talking about how to supply
oxygen for aerobic
institute by remediation i was briefly
talking about both active and passive
um you can actively pump hydrogen
peroxide and you can actively form
aerated
water into the system and passively you
can do
um you know pump air but
as i said it depends on where the
contaminant is you can do between
bio venting and air sparging by a
venting
is above the above the
the water system and as forging is below
if the oil is below
so it depends on the site yeah okay and
it's
uh it's the second one
all right what is the best method into
the bacteria consultation to degrade
biomaterial base
if they need different conditions should
we manually add
them one by one or we add them together
in the beginning
well it uh as i said it's uh it's um
you need to do a little bit of lab study
first uh
sometimes the nature throws some
surprises like i
showed you the last experiment we did
and the mixed condition worked we put
them all together that worked
better we didn't expect it at all so we
thought individual
electronic software worked better so
always uh
it's good to do a little bit of bench
scale study before
you can apply this so there is no one
uh size that will fit for all the
scenarios so i
i depend on the side it depends on what
biomaterial um you're trying to clean up
so okay so the
uh third one um the
effect that dissolved oxygen ammonia
levels on the growth antigen the
bacteria and vitamins are combined by
crude oil
the effect of dissolved oxygen ammonia
level
um we i didn't show you the the lab
study the
media and all that so you
when we did this um oil spill study
the aerobic conditions uh
yes oxygen really worked well
for our condition need to have anywhere
from
uh 2 milligrams per liter from 0.2 to 2
milligrams so we have like micro
aerophyllic to aero
big conditions so 2 milligram or less is
okay
oxygen in the water and then ammonia you
need to have
uh in our system 30 to 1 ratio
a carbon nitrogen ratio for every
30 parts of carbon um one part of
nitrogen so 30 to 1 ratio is what we
used in our study so um
so you got to do first how much nitrogen
is already there
in the site and then you work with that
carbon nitrogen ratio
any any other questions
i'm still mute so okay is there any
question for me if you have any
uh please follow me
yes uh thank you
i think yeah
ready yeah yeah okay uh right
yeah yeah uh kind of flow out in
indonesia
not too big somewhere near karawaka
yeah and they managed uh to
get the the field uh stop
but we do have a problem with mangrove
uh-huh
many of uh the plants were
[Music]
still contain some splashy oil
okay what's the best way to
secure the plant i mean
it's not possible to people because
mangrove is very
long to grow and it's kind of a pity
that they contaminated with
the oil sludge yeah any any idea
how to treat plants
so is a mangrove is planted still alive
they're not dead by this uh oil
sludge some of them they
still like yeah okay one that's
a badly yeah we have to get rid of them
but we still have some that uh
possibly they they will surprise that
i think they have to be
so in our experience do you do you have
some
um salt marsh also in the mangroves
you don't have any salt grass in that
area
because salt marshes really take up this
oil
very well so um and if you have salt
moisture i don't know whether you can
plant that will
enhance your oil degradation process
and and also it will promote the
rhizospheric
bacteria and so you have that
the site i showed you has some short
marsh
and we the sample that we collected the
root of those salt marks is
where this bacterial activity are
and those are the one that was actually
doing the oil
cleanup so so you can't have some
you know help from the nature um
otherwise you got it you got to add them
uh maybe promote some particular
anaerobic bacteria what is the redux in
the sediment
you know what kind of redux conditions
it's a wave action you have to take into
account the wave action
every time wave comes in the tidal
action
uh it will oxygenate your sediment
so yeah if it is kind of the zone
between
aerobic and nitro reducing then maybe
you got to promote that
activities there yeah okay we don't know
yet
yeah yeah and they'll have a lot of
those um
things you need to look into it but you
cannot just show
yeah this bacteria from the literature
seem to remove
maybe we have to do the no you have to
look in your site okay what kind of
plants are there what kind of redux
condition is there
how much wave action in your particular
site
is it a shallow coastal area
yeah yeah okay okay
yeah yeah yeah
okay yeah i mean
uh it is then when we start uh
discussing about that then we have a
funding so we haven't done anything
okay so you are involved in that project
so you got to collect some
treatability study collect some sample
looking what can activity you have
in the sediments mangrove samples
just to find out to what extent uh the
the disrespect to the plants
yeah it's largest in the in the
sediment of the mangroves and you can
look at the bacterial activity in that
sediment
whether but maybe sulfate reduces are
there if you if it smells hydrogen
sulfide definitely there is sulfate
reducing activity going on
so far we focus on the mangrove itself
mangrove itself yeah but he said the
mangroves are completely covered and so
yeah and then you need to have other
plants to help the mangrove so mangroves
are very very susceptible
to oil yeah very sensitive so
for oil yeah
thank you thank you so i think so
uh this one
for me i think the last one it might be
so raj uh
you mentioned about the uh
uh this station so i missed the leaking
from the
uh gas station yeah underground yeah
so that one the gas station
or the owner of the gas station cannot
afford to to pay
for the cleanup right
if the owner is still in business they
have to they have to do the cleanup
yeah but
you cannot find the owner then the
government will do the cleanup
oh the government will
clean up and then they have what you
call it they have a super fund
super fund yeah yeah in epa every
budget every year the government put the
money
in the budget called super fund and the
money is used for cleaning up the
priority site this there are
every year 1300
sites are considered priority site
so they update every budget year what
are the sites
need to be cleaned up first so they use
that super fund to clean up those sites
and and then they try to recover the
money later but
if the gas station guys are still in
operation then
the government will come and ask them
you know to clean up but if they're not
there they're gone
and nobody knows where they are then the
government you said
yeah yeah okay then
no no other questions is it's already
yes there are no other questions okay
i think we we may close our
uh class our session today and then
we i will for the next weeks so
uh because you mentioned uh the it might
be the topic on the micro bio leave it
on bio
next week is a biological treatment of
wastewater
okay so it's of your vertical treatment
and then the week after is
uh microbial lipids and lignocellulosic
okay okay next second biological
treatment
okay i'll talk about some basics and
then i'll do some case studies
okay things are
uh will you be close
uh i thought we can stop the uh
in in the youtube i would like to thank
and then
uh next week uh we will have
uh lecture from rosh on the biological
treatment
uh of wastewater thanks for
thank you all right so thank you
say hello to ross