Note: Descriptions are shown in the official language in which they were submitted.
CA 02229761 1998-02-17
BIODEGRADATION OF OIL SLUDGE
The present invention is directed to the treatment
of oil sludges, and in particular to biodegradation of
oil sludges to environmentally-acceptable products. As
such, the present invention is directed to the treatment
of compositions with high sludge/total petroleum
hydrocarbon concentrations, examples of which are oil
refinery sludgej, tank-bottom sludges from oil storage
tanks or tankers, sludges from residues at oil wells, so-
called slop oil or treater emulsions, oil sludges from
processing of solids containing oil wastes including
centrifuged sludges, clay fines, and drilling mud
residues. In contrast to waste water treatment processes
utilizing low total petroleum hydrocarbon concentrations
or processes for the production of single cell protein,
biomass or bacterial cells.
Biodegradation of crude oil materials has primarily
been directed to the clean up i.e. bioremediation, of
oil-contaminated soils and shorelines, as a result of on-
land oil spills from, for example, underground storage
tanks, or from oil tankers at sea. Such bioremediation
of hydrocarbons generally involves creation of conditions
in the soil or on the shoreline that promote growth of
microorganisms using the petroleum hydrocarbons,
facilitating conversion of the hydrocarbons to biomass
and/or their degradation, ultimately to carbon dioxide
and water. The hydrocarbons are the source of carbon for
microbial growth, although it may be necessary to add
other ingredients, especially nitrogen and phosphorus, as
fertilizers. Microorganisms also require a range of
inorganic ions for growth, but such ions are generally
present in adequate quantities in the soil that is being
treated.
CA 02229761 1998-02-17
2
Bioremediation processes generally utilize aerobic
microorganisms that require aeration/oxygenation by
maximizing contact of the contaminated material with
atmospheric oxygen through procedures of soil tilling or
by aerating using positive or negative pressure air
pumping systems.
The general hierarchy of microbial activity in crude
oil is understood to be
aliphatics>aromatics>resins>asphaltenes.
Thus, aromatic and high molecular weight hydrocarbons are
more difficult to degrade, compared to the lower alkanes.
Liquid-solid treatment systems have also been used
to degrade petroleum hydrocarbons. However, long
degradation treatment periods were encountered, e.g. 50-
100 days. Land treatment of waste crude oils and
refinery oil sludges has been used for many years as a
method of disposal of oil and sludge. Microbial growth
and biodegradation rates tend to be suboptimal in land
farming processes and the process is not easily
controlled. In addition the process is influenced by
soil composition, weather and temperature, as well as the
methods used for tilling in the land farming process.
For large refineries, large areas of land have to be
committed to such a process, and moreover the first step
in the process involves contamination of the soil with
the oils to be degraded.
U.S. 3 699 376 discloses a single or multi-tank
system that is primarily directed to waste water
treatment. The process utilizes a particular bacterial
strain from a culture collection for the bioremediation
process.
CA 02229761 2000-09-14
3
U.S. 5 364 789 discloses a microbial cleaner
comprising at least one hydrocarbon-ingesting microbe
strain and a biocatalyst that transforms hydrocarbons
into non-toxic substances. The biocatalyst includes a
non-ionic sur:=actant, a chlorine-absorbing salt, at least
one microbe nutrient and water. It is stated that the
cleaner may bE~ used in virtually any situation requiring
the removal oi= hydrocarbons, including cleaning
contaminated soil and treating oil spills on soil and
water.
A method for the biodegradation of a petroleum
hydrocarbon s=Ludge :fraction has now been found, such
method using a reactor.
Accordingly, an aspect of the present invention
provides a met: hod for the biodegradation of an oil-based
sludge, said oil-based sludge comprising a mixture of
petroleum hydrocarbons, said method comprising the steps
of
(a) forming an aqueous solution in a reactor of an
oil-in-water emulsion, bacterial culture and nutrients
for said bacterial culture,
said oil--in-water emulsion being an emulsion of said
oil-based sludge in water,
said bacterial culture having the ability to grow on
petroleum hydrocarbons as sole carbon source and having
been isolated from a hydrocarbon contaminated soil or
hydrocarbon-containing sludge or other environments rich
in hydrocarbon degrading bacteria, by microbial
enrichment tec:hniquE~s using hydrocarbons in the selection
medium,
said reacaor containing up to 50% by volume of
petroleum hydrocarbons;
CA 02229761 2000-09-14
4
(b) maintaining said aqueous solution under aerobic
conditions in the reactor at a temperature of at least
10°C for a period of time sufficient to reduce the amount
of petroleum :hydrocarbons by at least 25%, and at a pH
conducive for promotion of bacterial growth and
hydrocarbon d~=gradation, said bacterial culture growing
on said petroleum hydrocarbons and thereby reducing the
amount of petroleum hydrocarbons by intracellular
metabolism thereof; and
(c) discharging aqueous solution having a reduced
amount of petroleum hydrocarbons from said reactor.
In a pre:Eerred embodiment of the present invention,
the nutrients comprise bioavailable nitrogen, phosphorous
and potassium compounds, especially in which the nitrogen
compound is an ammonium ion, nitrate or organic nitrogen,
and the phosphorus is phosphate.
In anothE~r embodiment, the reactor contains about 5-
50% by volume of said petroleum hydrocarbons, especially
about 10-30% by volume of said petroleum hydrocarbons.
The oil-based sludge preferably contains
hexane-extract:able ;hydrocarbons in an amount in the range
of up to 500 000 ppm, especially in the range of 65 000-
250 000 ppm.
In yet another embodiment, the nutrients are in
proportions corresponding to relative proportions in
bacterial cel='~s, and supplied at concentrations which
- promote high _~evels of bacterial growth and high rates of
hydrocarbon dE:gradation.
In further embodiments, the petroleum hydrocarbons
consist of mixtures of saturated hydrocarbons, aromatic
hydrocarbons, hydrocarbon resins and asphaltenes,
especially petroleum hydrocarbons obtained from petroleum
refinery sludge, from the bottom of a storage tank for
CA 02229761 2000-09-14
oil, from an on-land well head or from the washing of a
hold in a tanlter .
In other embodiments, the amount of nitrogen
5 required to support the process is in the range of 50-
1000 ppm, and preferably in the range of 300-700 ppm, and
the minimum amount of phosphate is in the range of 10-200
ppm and prefe=rably 50-150 ppm.
In addit=ional embodiments, the aqueous solution
contains a surfacta=nt, more especially a non-ionic or an
anionic surfactant. The surfactant is in an amount
sufficient to form aaid oil-in-water emulsion, especially
in which the amount of surfactant is less than 2500 ppm
and preferably less than 1500 ppm. It is preferred that
the ratio of t;he amount of petroleum hydrocarbon to
surfactant be at least 40:1.
In furthe=r embodiments, the amount of petroleum
hydrocarbons ~:s reduced by at least 50% and especially at
least 75%.
In other embod=iments, the sludge is admixed with
organic molecules ol:her than petroleum hydrocarbons. In
step (b), the organ=ic molecules may or may not be
degraded.
A further- aspect of the present invention provides a
method for the' biodegradation of an oil--based sludge,
said oil-based sludc3e comprising a mixture of petroleum
hydrocarbons, said method comprising the steps of:
- (a) forming an aqueous solution in a reactor of an
oil-in-water emulsion, bacterial culture and nutrients
for said bacterial culture,
said oil--in-war:er emulsion being an emulsion of said
oil-based sludge in water,
said bact=erial culture having the ability to grow on
petroleum hydz-ocarbons as sole carbon source
CA 02229761 2000-09-14
5a
and having been isolated from a hydrocarbon contaminated
soil or hydrocarbon-containing sludge or other
environments :rich in hydrocarbon degrading bacteria, by
microbial enrichment techniques using hydrocarbons in the
selection medium,
said reactor containing up to 50% by volume of
petroleum hydrocarbons;
(b) maintaining said aqueous solution under aerobic
conditions in the reactor at a temperature of at least
10°C for a period of time sufficient to reduce the amount
of petroleum hydrocarbons by at least 25%, and at a pH
conducive for promotion of bacterial growth and
hydrocarbon dE~gradation, said bacterial culture growing
on said petroleum hydrocarbons and thereby reducing the
amount of petroleum hydrocarbons by intracellular
metabolism thereof; and
(c) discharging aqueous solution having a reduced
amount of petroleum hydrocarbons from said reactor.
The method of the present invention relates to the
biodegradation of an oil-based sludge. The oil-based
sludge compri;~es a mixture of petroleum hydrocarbons and
may include non-petroleum solid or liquid contaminants
and water. The petroleum hydrocarbon mixture would
normally comprise a mixture of aliphatic hydrocarbons,
aromatic hydrocarbons, hydrocarbon resins and
asphaltenes.
The present im,rention is particularly directed to
the biodegradation of a mixture of the petroleum
hydrocarbon from among the aliphatics, aromatics, resins
and asphalten~~s. Such mixtures of petroleum hydrocarbons
may be obtained from a variety of sources. For instance,
the mixture may be :in form of a sludge obtained from a
petroleum refinery. The sludge may also be obtained from
CA 02229761 2000-09-14
5b
the bottom of a storage tank that has been used for the
storage of petroleum oil, with the sludge being obtained
particularly when the storage tank is cleaned or drained.
S Alternatively, the mixture of hydrocarbons could be a
CA 02229761 1998-02-17
6
petroleum residue obtained from around an on-land well
head, be an oil-containing clay fines material or be
or from the cleaning of a hold of a tanker used for the
transportation of petroleum products. The mixture of
petroleum hydrocarbons, which is referred to herein as a
sludge, may also be obtained from a variety of other
sources. In each case, the sludge is characterized by
having a substantial proportion of heavy end petroleum
hydrocarbons which may require use of a solubilizing
agent or surfactant to facilitate mixing and dispersal in
water, as an oil-in-water emulsion.
The method of the present invention is carried out
in a reactor. It is preferred that the reactor be a
single stage reactor that is charged with the solution
described herein, allowed to incubate for a period of
time to reduce the amount of hydrocarbons within the
aqueous solution, and then subsequently discharged from
the reactor. Nonetheless, it is to be understood that
the reactor could be in the form of a series of reactors
in which the aqueous solution is passed from reactor to
reactor before being finally discharged from the process
for the biodegradation of the sludge.
In the method, an aqueous solution is fed to the
reactor. The aqueous solution is comprised of an oil-in
water emulsion, bacterial culture and nutrients for the
bacterial culture. The sludge is in the form of the oil-
in-water emulsion.
The amount of petroleum hydrocarbons fed to the
reactor is primarily governed by the formation of the
oil-in-water emulsion. In particular, the aqueous
solution may contain up to 50% by volume of petroleum
hydrocarbons, depending on the particular hydrocarbons,
CA 02229761 1998-02-17
7
or higher if the petroleum hydrocarbons will permit
formation of oil-in-water emulsions at higher loadings.
In preferred embodiments, the reactor contains 5-50% by
volume of the petroleum hydrocarbons, especially 10-30%
by volume.
The oil-based sludge contains hexane-extractable
hydrocarbons. In preferred embodiments, the amount of
hexane-extractable hydrocarbons is up to 500 000 ppm,
especially in the range of 65 000 - 250 000 ppm.
It would normally be necessary to incorporate a
surfactant into the aqueous solution and to subject the
aqueous solution to agitation in order to form the oil-
in-water emulsion. The surfactant is preferably a non-
ionic or an anionic surfactant, and is used in an amount
sufficient to form the emulsion. Nonetheless, the amount
of the surfactant is preferably less than 2500 and
particularly less than 1500 ppm. In addition, the amount
of surfactant, if added, is maintained at as low a level
as is consistent with obtaining the oil-in-water
emulsion. In particular, it is preferred that the ratio
of petroleum hydrocarbon to surfactant be at least 40:1,
and especially at least 60:1»
The aqueous solution also contains a bacterial
culture. The bacterial culture used in the method of the
present invention is a natural-occurring bacterial
culture. Such a culture may be isolated from a
hydrocarbon-contaminated soil or from hydrocarbons-
coataining sludge or from other environments, including
soil or activated sludge, which may be rich in
hydrocarbon-degrading bacteria, and inoculated in a basal
medium, as described herein. The bacterial culture is
selected by its ability to grow on petroleum hydrocarbons
CA 02229761 1998-02-17
8
as the predominant source of carbon in the basal medium.
Bacterial enrichment techniques for isolation of a
bacterial culture capable of growing on hydrocarbons are
well understood in the art. Typical techniques comprise
adding a sample of soil, sludge or other material
containing a large population of bacteria to an aqueous
medium containing hydrocarbons as the only or predominant
carbon source. Other chemical components including an
inorganic nitrogen source, phosphorous and salts
necessary to support bacterial growth are also added.
Such a medium can be used to preferentially promote
multiplication ~f hydrocarbon-degrading bacteria using
standard aerobic microbial cultivation methods, including
incubation in aerated microbial culture vessels. By
transfer of a small amount of the resultant growth
culture to further samples of the same medium and
repeating the process one or more times, an efficient
hydrocarbon degrading culture is selected. The culture
can be maintained or stored using methods well known in
the art.
In order to prepare a high density culture for use
as an inoculum for sludge degradation, the maintained
culture may be inoculated into an aqueous medium
consisting of the nutrients described herein,
supplemented with petroleum hydrocarbons and incubated in
an aerated reactor or fermenter or other culture vessel.
The preferred inoculum volume is 0.1-20% by volume of
total culture volume, preferably 1-5% by volume. The
preferred concentration of petroleum hydrocarbons used in
this inoculum development medium is 0.5-5$, and can be
obtained from various sources including petroleum
sludges, crude oils or refined oils such as diesel oil.
CA 02229761 1998-02-17
9
A typical aeration rate of the inoculum reactor is 0.1-
1.0 volumes of air per volume of medium per minute, with
the culture incubated in the temperature range 20-37°C for
1-7 days, preferably at 27-33°C, at a pH generally
maintained in the range 6.5-8.0, preferably in the range
7-7.5. The resultant bacterial culture maybe used to
inoculate the reactor containing the sludge to be
degraded, at a rate of 0.1-20% of total sludge volume,
preferably 1-5%. Where a much larger volume of inoculum
is required, the resultant inoculum may be transferred as
an inoculum to a larger culture vessel and the culture
development process repeated on the larger scale.
The aqueous solution fed to the reactor also
contains nutrients for the bacterial culture. A wide
variety of nutrients for the bacterial culture may be
used, as will be understood by persons skilled in the
art. Such nutrients will include nitrogen, phosphorus
and potassium compounds, and would normally also include
a variety of other ingredients. In particular, the
nutrients comprise bioavailable nitrogen and phosphorus
compounds. In embodiments, the amount of nitrogen is in
the range of 50-1000 ppm and preferably 400-700 ppm, and
the amount of phosphate is in the range of 10-200 ppm and
preferably 50-150 ppm. In addition to nitrogen and
phosphorus compounds, the nutrient also contains
optimized concentrations of compounds other than
nitrogen, phosphorus, carbon, oxygen and sodium, required
to support bacterial growth and therefore it is normally
necessary to add to the reactor one or more of magnesium,
manganese, inorganic or organic sulphur, calcium, iron,
copper, cobalt, zinc, boron and molybdenum. It will be
appreciated that a guide for selection of the relative
CA 02229761 1998-02-17
amounts of nitrogen, phosphorus and other required
nutrients is to relate their concentrations to the
amounts of these components present in bacterial cells.
By providing an appropriate balance of nutrients and
S by adjustment of nutrient concentration, it is possible
to achieve high levels of growth of hydrocarbon degrading
bacteria and thus accelerated rates of hydrocarbon
degradation. For example, Greasham (1993)
"Biotechnology, a multivolume comprehensive treatise"
10 (Eds, Rehm, H.J., et a1) Vol. 3, p.131, VCH, Weinheim)
has reported the typical non-carbon elemental composition
of major bacterial components to be nitrogen 12.5%;
phosphorus, 2.5%; potassium, 2.5%; sodium, 0.8%; sulphur,
0.6%; calcium, 0.6%; magnesium, 0.3%; copper, 0.02%;
manganese, 0.01% and iron, 0.01%. Use of appropriate
concentrations and ratios of nutrients tends to avoid a
situation where growth is limited by depletion of one
essential nutrient while all other nutrients may be
present in excess.
The hydrocarbon provides the carbon source for
growth; oxygen is obtained by aeration of the culture;
sodium is provided~in the form of caustic soda, required
to adjust the pH. It is also understood that in some
cases, some of these nutrient components may be present
in sufficient quantities in some petroleum sludges or
added water such that addition of selected nutrients may
in some cases not be required. A disadvantage of relying
on nutrients present as contaminants in sludge or water
is that their concentrations may be variable, thus
introducing inconsistencies into the process.
CA 02229761 1998-02-17
11
An example of a nutrient composition is as follows:
N as NHa, N03, or organic N 500-700 ppm
P ~.~ s phosphate or related form 100-120 ppm
K 50-90 ppm
Mg 10 ppm
Mn 1-4 ppm
S as sulphate or organic sulphur 15 ppm
Ca 8-12 ppm
Ferric Ion 1 ppm
Copper 0.5 ppm
Surfactant (nonionic or anionic) 1250 ppm
Co, Zn, B, Mo S-10 ppb each
The relative ratios of these nutrients are similar
to the ratios typically found in the compositions of
bacterial cells.
Other examples of nutrient compositions are given in
the Examples herein below.
The aqueous solution in the reactor is maintained at
a temperature of a least 10°C. Preferred temperatures are
15-37°C, and especially 20-33°C. The aqueous solution is
maintained in the reactor for a period of time sufficient
to reduce the amount of total petroleum hydrocarbon by at
least 25%, especially by at least 50%. Typical times to
effect the reduction in total petroleum hydrocarbon is 5-
20 days, depending on the petroleum hydrocarbon being
treated and the reactor conditions.
Subsequent to maintaining the aqueous solution at
the predetermined temperature for a period of time, the
aqueous solution is discharged from the reactor. The
aqueous solution has a reduced amount of hydrocarbons,
CA 02229761 1998-02-17
12
including a reduced amount of the hydrocarbons from the
group comprising the aromatics, resins and asphaltenes.
The present invention may be used for the
biodegradation of sludges, as described herein. In
particular, it may be used for biodegradation of a
combination of hydrocarbon components from among the
fractions: saturates, aromatics, resins and asphaltenes.
It may also be used to preferentially degrade a
proportion of the hydrocarbons, in a manner which causes
the emulsion to break and facilitate separation of a
water phase and a residual oil phase. The residual oil
phase may be recovered for reuse. Alternatively, the oil
phase may be recycled to the next reactor cycle with the
water phase only being discharged from the reactor. The
water phase contains high concentrations of hydrocarbon-
degrading bacteria. Thus, the water phase may be used
for processes including soil bioremediation processes, by
direct spraying of the water on the contaminated soil.
Alternatively, the bacteria maybe recovered from the
water phase by known methods (filtration or
centrifugation) and subsequently the bacteria may be
applied in these other processes.
Where subsequent batches of sludge are to be
degraded in the reactor, a portion of the degraded sludge
amounting for example, to 1-20% of reactor volume, may be
retained in the reactor following discharge, as an
~ inoculum source for the next sludge batch.
In addition to the above described batch sludge
degradation process, it is envisaged that the invention
extends to fed-batch, continuous and semi-continuous
reactor processes. In the fed-batch process, after the
batch process has proceeded for some time, additional
CA 02229761 1998-02-17
13
sludge and/or nutrients/surfactant are added at one or
more intervals and the process is allowed to continue.
In continuous or semi-continuous processes, degraded
sludge is removed from the reactor and replaced with
undegraded sludge and nutrients/surfactants on a
continuous basis or at intervals, respectively.
The invention is illustrated by the following
examples. Unless stated to the contrary, all examples of
the invention illustrated herein were conducted under
non-sterile conditions. In addition, all biodegradation
reactions exemplified herein used oil-in-water emulsions.
waunr r r
The basal medium used in this example contained (per
L) : KH2P0" 1.0 g; NaZHPO" 1.5 g; MgS0,.7H~0, 0.2 g; NaZC03,
0.1 g: CaCIZ.2H20, 0.05 g: FeSO,, 0.005 g; MnSO" 0.02 g;
and trace metal solution, 3 ml. The trace metal solution
contained (per L) : ZnClz.4Hz0, 0.0144 g; CoClz, 0.012 g;
Na2Mo0,.2H20, 0.012 g; CuS0,,5H20, 1.9 g: H3B0" 0.05 g: and
HC1, 35 ml. The initial pH of the nutrient was adjusted
to 7.2.
A population of mixed bacterial culture was
maintained in a cyclone fermenter with a working volume
of one litre. Petroleum hydrocarbon-degrading bacteria
were selected by their ability to grow on petroleum
hydrocarbons as the sole carbon source in the basal
medium described above. To initiate the selection of
petroleum hydrocarbon-degrading bacterial culture, a
mixed population of bacteria, isolated from hydrocarbon
contaminated soil, was inoculated into basal medium
supplemented with 2.0 g NH,C1/L and 1.0 g NaN03/L in the
cyclone. Sludge A or B (50 g/L) was used as carbon
CA 02229761 1998-02-17
14
source; the sludges are describe below. It was found
that the bacterial population reached 10° to 101° CFU/ml
in one week. Thereafter, the culture was maintained by
removing 10% by volume of the reactor and replacing with
10% by volume of fresh basal medium and sludge every day.
Using this procedure, an actively growing culture was
maintained.
Sludge samples were collected from different ponds
or lagoons located at different oil refineries. TPH
content (hexane extractable) was determined for each
sludge. The composition of the different sludges is
provided in Table 1.
Table 1.
Sludge Hexane Hexane Water
Source Soluble(%) insoluble(%) (%)
Sludge A 25 13 62
Sludge B 13 3 84
Sludge C 12 11 77
Sludge D 65 15 20
Sludge E 31 16 53
Sludge F 22 6 72
Sludge G 89 ~ 11 0
EXAMPLE II
The nutrient medium used for biodegradation in this
example consisted of the basal medium supplemented with
2.0 g urea/L and 1.0 g yeast extract/L.
Runs to determine the biodegradation of total
petroleum hydrocarbons (TPH) with respect to incubation
time were carried out in 250 ml Erlenmeyer flasks
containing 10 ml of nutrient medium and 10 g of sludge,
giving a final sludge concentration of SO% in the total
flask contents. The flasks were inoculated with 0.6 ml
CA 02229761 1998-02-17
of actively growing mixed culture from the cyclone,
maintained as described above, and incubated for 24 days
at 25°C.
Residual TPH content was determined as follows. At
5 different time intervals, whole flask contents were
extracted with 40 ml of hexane and centrifuged at 10 000
rpm for 20 minutes. The hexane layer (top) was pipetted
out and transferred to a pre-weighed vial. The hexane
was allowed to evaporate in a fumehood and residual oil
10 was weighed to determine total petroleum hydrocarbons
(TPH) .
The results are given in Table 2.
Table 2.
Incubation Time (Days) TPH degradation ($)
6 32
14 37
18 47
24 4B
It was found that over a period of 18 days, about
47$ degradation of TPH occurred. No significant
difference in degradation levels was obtained between 18
days and 24 days.
EXAMPLE III
In order to investigate the effect of surfactant on
TPH biodegradation, 5 different surfactants were tested
at 0.25$ concentration.
In each test, 10 ml of nutrient medium, 10 g of
sludge oil and 0.5 ml of stock surfactant solution (10%
aqueous) were placed in a 250 ml Erlenmeyer flask. The
CA 02229761 1998-02-17
16
contents of the flask were inoculated with 0.6 ml of
actively growing culture from a cyclone fermenter and
incubated on a rotary shaker (200 rpm) for 14 days at
25°C. Residual TPH content was determined after
S extraction with hexane.
The results are given in Table 3.
Table 3.
Surfactant TPH degradation (%)
None 46
Igepaln' CO-630 66
Biosoft~' EN-600 63
Sorbax~' PM030 42
Witcomul~' 4078 41
Marlipal~' 0 13/120 45
All surfactants gave an oil-in-water emulsion. Out
of 5 surfactants tested, 2 surfactants viz. Igepal CO-630
and Biosoft EN600, were found to be more effective.
About 66$ degradation of TPH was achieved in the presence
of the Igepal surfactant, compared to 46% in a control
run in the absence of any surfactant.
EXAMPLE IV
The effect of sludge concentration on TPH
biodegradation was investigated using two different
sludges at concentrations of 20%, 50~ and 90%. Each set
of flasks contained the following: (a) 16 ml of nutrient
medium and 4 g of sludge; (b) 10 ml of nutrient medium
and 10 g of sludge; (c) 20 g of sludge and 2 ml of lOx
strength nutrient medium. The flasks (250 ml) were
CA 02229761 1998-02-17
17
inoculated with 600 ~1 of actively growing culture from a
cyclone fermenter, and incubated on a rotary shaker (200
rpm) at 25°C for 14 days.
The results are given in Table 4.
Table 4.
Sludge Type Sludge Concentration TPH degradation
% v/v (% of
starting amount)
Sludge A (a) 20 70
(b) 50 56'
(c) 90 36
Sludge B (a) 20 91
(b) 50 B1
(c) 90 56
It was found that sludge concentration affected the
extent of TPH degradation.
V ..
A medium referred to herein as NPK medium was formed
by replacing, KHZPO, and NaZHPO" in the nutrient medium,
were replaced with.a NPK (nitrogen: phosphorus: potassium)
fertilizer (15:30:15) at a rate of 0.8 g/L. All other
components in the medium were the same as described
before. Experiments were conducted with two different
sludges. Erlenmeyer flasks contained 50% v/v NPK medium
and 50~ v/v sludge together with 0.25% surfactant (Igepal
CO-630) based on total culture volume.
Other conditions were the same as those described in
Example III. The results are given in Table 5.
. s . .
CA 02229761 1998-02-17
18
Table 5.
Source of Sludge Medium TPH degradation
(% of starting amount)
Sludge A Nutrient 60
NPK 58
Sludge B Nutrient 73
NPK 71
No significant differences were observed between the
results obtained with basal medium and with NPK medium.
EX1~I~I~E VI
Biodegradation of TPH in different sludges was
performed in flasks under shaking conditions. Erlenmeyer
flasks containing NPK medium and sludge (50:50, v/v) were
inoculated with the actively growing mixed culture, and
incubated for 14 days at 30°C.
Table 6.
Sludge Type Sludge Concentration(%) TPH degradation (%)
A 50 61
B 50 76
D 12.5 54
E 50 89
G 10 42
The results indicate that 42 to 89% degradation of
TPH can be obtained using this process. Sludge G, being
a heavy oil sludge, was degraded the least.
~.~. -. . r
CA 02229761 1998-02-17
19
EXAI~LE VII
Alternative complex nitrogen sources to yeast
extract were tested using Sludge A and Sludge B. This
experiment was carried out using NPK medium 50% v/v,
sludge 50% v/v and 0.25% v/v Igepal CO-630 in 250 ml
Erlenmeyer flasks incubated at 25°C for 14 days on a
rotary shaker (200 rpm).
The results are given in Table 7.
Table 7.
Sludge Complex TPH
Type nitrogen degradation
source (% of
starting
amount)
Sludge A Yeast extract 59
Corn steep solids 52
Cottonseed protein 51
Potato protein 49
Sludge B Yeast extract 75
Corn steep solids 85
Cottonseed protein 83
Potato protein 79
All the alternative nitrogen sources tested, at a
final culture concentration of 0.5 g/L, gave similar
performance to 0.5 g/L yeast extract.
EXAMPLE VIII
Biodegradation of different hydrocarbon fractions
was tested, using Sludge B. Erlenmeyer flasks that
contained 50% v/v sludge, 50% NPK medium and 0.25% Igepal
C0630. After inoculation with an actively growing
culture, flasks were incubated on a rotary shaker for 14
days at 30°C. The whole content of the flask was
CA 02229761 1998-02-17
extracted once with hexane followed by dichloromethane.
After centrifugation both extracts were combined and the
solvent evaporated. Residual hydrocarbon was dissolved
in hexane and centrifuged. A known weight of hexane
5 soluble portion was passed through a column (0.75 x 27
cm) of silica gel (activated at 100°C overnight).
Successive ,applications of hexane (120 ml),
dichloromethane (30 ml) and chloroform: methanol (1:1, 15
ml) produced fractions containing saturated, aromatics
10 and polar (resins) hydrocarbons, respectively.
The results are given in Table 8.
Table 8.
15 Fraction % of total
hydrocarbons % degradation
Saturate 73-77 73-77
20 Aromatics 11-13 65-69
Resins 8-10 61-63
The results indicate that all of the major TPH
component were degraded.
EXAMPLE IX
This experiment was conducted to determine if
pretreatment with an advanced oxidative process (Fenton's
reagent viz. H202 + FeSO,) could enhance TPH degradation
in sludge. Pretreatment and subsequent biodegradation
was carried out in the same flask. For pre-treatment,
Sludge A was diluted with water to obtain 20 ml of a 50%
v/v sludge concentration. pH of the mixture was adjusted
to 4.0 by adding 4N HC1. H202 and FeSO, were added at
concentrations of 0.3$ v/v and 10 millimolar,
respectively. -
CA 02229761 1998-02-17
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The flasks were kept on a rotary shaker (200 rpm) at
25°C for 2 days. Thereafter, 2 ml of NPK medium (10 times
concentrated) were added in solid form and the pH
adjusted to 7.0 by addition of 2N NaOH solution. The
flasks were inoculated with an actively growing inoculum
(600 ~,1) from a cyclone fermenter and incubated on a
rotary shaker for a period of 28 days. The following
treatments were tested: (a) no pre-treatment or addition
of surfactant; (b) Fenton's reagent pre-treatment,
without surfactant; (c) addition of 0.25% Igepal CO-630,
without Fenton's pretreatment: and (d) Fenton's reagent
pretreatment in the presence of 0.25% Igepal CO-630.
The results are given in Table 9.
Table 9.
Treatment Incubation time (days)
7 14 21 28
% TPH degradation
None 28 42 46 53
Fenton's pre-treatment
(48 h) 36 61 64 65
Surfactant (0.25 %) 42 61 66 72
Fenton's pre-treatment
(48 h) in the presence of
surfactant (0.25 %) 43 64 68 72
The results indicate that pre-treatment of sludges
with an oxidative agent or addition of surfactant
significantly increased the extent of degradation of TPH
in sludge oil.
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EXAMPLE X
The performance of biodegradation of TPH in
different sludges was evaluated in different reactor
types. The reactors tested were of different
configuration and scale. Biodegradation tests in
Erlenmeyer flasks were performed as described in other
examples. Cyclone fermenters were as described above.
Air-lift reactors were fitted with spargers and connected
to an air source. The mixing in the reactors was achieved
by supplying air at the rate of 0.5 volume/volume/minute
and 0.125% surfactant. NPK medium and sludge (50:50 v/v)
was used in these experiments. All the reactors were
inoculated with an actively growing mixed culture.
The results are presented in Table 10.
Table 10.
Sludge Reactor Scale of Incubation TPH
Type Type Process time biodegradation
(litres) (days) (%)
E Erlenmeyer 0.25 20 74
flask
E Air-lift 150 14 70
F Air-lift ~~ 150 14 84
B Erlenmeyer 0.25 14 81
flask
B Cyclone 1 8 B5
C Air-lift 18 000 11 84
The results show that efficient sludge degradation
occurs in different aerated reactor types and at
different scales of operation ranging from laboratory to
production scale.
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EXAMPLE RI
Biodegradation of TPHs in clay fines was evaluated
in shake flask cultures. Flasks containing clay fines
(TPH, 10.5%, w/v, and NPK medium 50:50, w/v) and 0.25%,
w/v Igopal CO-630 were inoculated with an actively
growing culture and incubated for 14 days at 30°C. The
residual TPH content was determined and results are shown
in Table 11.
Table 11
Incubation Time % TPH degradation
(days)
7 77
14 92
The results indicate that 92% of clay fines can be
achieved in 14 days by using this process.
