Note: Descriptions are shown in the official language in which they were submitted.
~L~
BACTERIAL COMYOSITION FOR RECOVERY OF OIL-
PO~IUTED WATER A~D SOIL
The pre~ent invention relate~ to environmental protec-
tion, refers to biological recovery of oil-polluted water
and soil and, more particularly, to a bacterial compo~i~ion
intended for this specific purpose.
The wide-spread pollution of water and ~oil with oil
component~ observed at present i8 attributed to the ever-
growing volumes of output9 transportation, refining and uti-
lization o~ oil and oil products. Such traditional methods
of recovery aR mechanical, chemical and phyaical fail to
provide a sufficient degree o~ recovery of oil-polluted
water and 80ilo ~herefore, it has been suggested to achie~e
this aim by resorting to the resources of Mature itself9
i.e. the effect o~ microorganisms capable of assimilating
the hydrocarbons of oil. It ha~ been e~tablished by research
thst in the oil spilled over the sea surface in the form
of a film 0.1 - 0.4~um thick the nonvolatalized components
of oil are attacked by various microbes which degrade a con-
siderable proportion of oil within 2 - 3 months. Besides,
it i8 emphasi~ed by ~cientific re~earch that the amount of
oil decomposed by other orgsnisms or by natural o~ida-tion
amounts to one tenth the amount degraded by microbes.
~ owever, in case o~ heavy pollutions, the process of
natural 0elf-recovery of water basins may stretch over a
long time, reaching several scores of ye~rs. It has bee~ cal-
culated that the rate of biodegradation i~ 0.5 kg~ha/day
2 --
so that degradation of 64 00 t of oil spilled after a
tanker wreck may take as long as 20 years approxi-
mately. (Microbial hydrocarbon degradation within
intertidal zones impacted by the Amoco Cadiz oil
spillage. Atlas R.M., Bronner A. "Amoco Cadiz.
Consequences pollut. accident. hydrocarbons. Actes
Collog. Int., Brest, 19.22 Nov. 1977 Paris, 1981, pp.
251-256).
Known in the prior art are methods for
removal of oil from the water surface by the use of
microorganisms and stimulating their activity by
introducing the sources of nitrogen and phosphorus
into the polluted medium (US Patent No. 4042495, US
Patent No. 4087356). However, the population density
of microorganisms of natural biocenoses is so low
that even intensive mineral feeding failed to bring
about the satisfactory degree and time of oil
degradation on water.
A more promising proposition in this field
was constituted by application to the polluted area
of bacterial cells of -the hydrocarbon-oxidizing
microorganisms either in the form of pure isolated
cultures or a combination of several genera and
species of said microorganisms (Microbiological J.
47, No. 2, publ. 1985, E.I. Kvasnikov, T.M.
Klushnikova, S.L. Kuberskaya, V.S. Zalevsky, G.F.
Smirnova, T.P. Kasatkina, V.I. Svarnik, A.A. Koval
"The use of bacteria associations for recovery of
oil-polluted bilge water", pp. 12-14).
The difficulties of controlling the
quantitative and qualitative composi-tion of bacterial
associations denied the possibility of launching the
industrial employment of this
3 --
method.
The wide~t recognition wa~ given to the use o~ micro-
organi~m~ mutants produced by the gene engineering methoda.
Such "~upermicrobe~" exhibiting a hlgh oil-oxidizing
activity are capable o~ degrading amall amount~ of spilled
oil in a ~hort time but fail in cases o~ massive pollutions
which call for longer period~ o~-oil degradation. Thua9 it
i8 known, that the life apan of the mutant, genus Pseudomonas,
i8 but 7.5 days from the ~tart of multiplication of desth,
which limits its employment only to a cxude oil content in
water not exceeding 50~ mg~1 (European Patent No. 000774223
IPC Cl2 ~ 1/209 Sept. 26, 1984). In addition9 it has been
~ound that the mutants are un~-tsble and apt to lo~e quickl~
the properties inoculated to them in natural conditions.
The pure cultures i~olated from the natural envlronment
prove to have more stable propertie~. ~or example, there iB
a known ~train Pseudomonas putida-36,~eparated from an oil-
polluted ares of 80il that has remained ~n~ecovered for a
long time; said strain i~ depo~ited under No~ B-2443 in
the Central Museum of Industrial Microorganisms of the Re-
search Institute "VNIIGENETIKA". Thie ~train i~ utilized for
the recovery o~ oil-polluted water and soil (USSR InYentor'~
Certificate ~o. 1076446, IPC C12 N 15/00, February 28~ 84),
The prolo~ged e~istence of the strRin under extreme con-
dition~ ha~ adapted it perfectly not only to B high degree
of oil pollution but alao to the preaence of highly-to~ic
chemic~ls in the habitat ~nd to ~harp variation~ of tempera-
ture. ~he high resi~tance of the atrain and the ctability
of its oxidizing properties made it useful in
recovery of natural fresh and sea-water basins,
industrial sewages of oil refineries, oil storage and
transport tanks and oil-polluted soils.
Ilowever, the degree of water and soil
recovery from various kinds of oil remained
insufficient, being 68.4% for aromatic and 74.5% for
paraffin oils.
*hus, the problem of removal of oil and
oil products from the surface of water and soil
remains pressingly urgent and calls for elaboration
of new, more efficient methods and means.
An object of -the present invention is to
provide a bacterial composition for the recovery of
oil-polluted water and soil which would provide for
the highest degree of recovery while being cheap,
readily available and simple in preparation and
industrial utilization.
The above object has been achieved by
developing a bacterial composition for the recovery
of oil-polluted water and soil, the active biological
componen-t of said composition being the strain
Pseudomonas putida-36 deposited under No. B-2443
which, according to the invention, is characterized
in that its mineral component is a mixture of mineral
salts in the following proportions, mass %:
KN03 34.26 - 37.12
NH4C1 28.66 - 31.28
NH4H2P04 25.42 - 28.71
NH4N03 2.89 - 11.66
The claimed composition makes for a
maximum elimination
-- 5 --
of polluting oil from water and ~oilO
A good practice i~ to u~e the ~train Pseudomona~ pu-
tida-36 containing from 1.8 . 101 to 3.6 . 1012 living
cells per g of dry biomass.
The composition containin~ the above strain with
1.8 101 to 3.6 . 1012 living cells per g of dry bio-
mass proves to be most effective.
It i8 practicable that the maos rBtiO in ~aid composi-
tion between the biocomponent and mineral component~ i.e~
the mixture of said ~alts VJOUld be 1:26 - 32.
The above ratio i~ ~uf~icient for maintaining the ac-
tive vitalit~ of said strai~.
The claimed composition may be used with or without
a carrier, solid or liquid one. Water may be used as a li-
quid carrier while talc, diatomite, kaoline, paraffin, etc.
as a ~olid one,
~ he content of said strain in the compo~ition with li-
quid (water) carrier i~ 104 to 106 cells/ml and that o~
~he mi~ture of ~aid salts, 0.07 - 0~08 mas~ %O ~hi~ amount
of the biocomponent is mo~t rational because the content of
biocomponent cells below 104 will prove ineufficient for
normal growth and development of cells while the content
of the mixture o~ said salt~ below .07 mas~ ~0 at the gi~en
content of biocomponent cells will be insuf~icient for the
normal growth of the biocomponent; at the same time, the
content of biocomponent cells exceeding 106 i~ inexpedient
~ince in presence of hydrocarbon~ in oil pollution~ their
content increa~es due to natural multiplic~tion.
~ ~æ~
-- 6 --
An objeot of the invention al~o lie~ in providing a
method of recovery of oil-polluted water and ~oil compri~-
ing the use of said compo~ition without a carrier, applied
at the rate of 350 - 800 mg/m2 of the polluted area~
~ hlB amount of the COmposition is enough for an all-
round recovery of water polluted by up to 25 kg/m3 of oil
and of ~oil containing up to 10 kg/m2 of oil. -
~ he polluting agents in said methods may be crude oiland its products such aa mazouts, lubricants, oils, fuels.
The claimed invention improves the recovery of oil-
polluted water and soil by 22 - 32 % a~ compared with the
use of the strain covered by the USSR I~ventor's Certificate
No. 1076446 and permit~ con~erting the hydrocarbons to the
eeologically neutral compound~ thus attaining a high en-
vironmental effect.
The claimed composition iB innocuous to man and e~vir-
onment being active both in fresh and sea water and on land.
~ he composition features a high resi~tance to chemic-
al pollution of water and soil with toxic elements of dril-
ling muds and other chemical reagent~.
The~e and other advantage~ of the invention will be-
come apparent from the detailed de~cription that follows.
The strain Pseudomonas putida-36 used in the composi-
tion iB isolated from the wa~te drilling mud on organic ba~-
iR (oil) in the Tyumen oil fields, USSR, and deposited und-
er ~o. I~M n M B-2443 in the Central ~useum of Industrial
microorgani~ms of the Research Institute "VNIIGE~ETIKA".
~ he Pseudomonas putida-~6 strain has the following
-- 7
characteriatic~
Morpholo~ical and culture_ propertie~
Size of one-day culture ~ 1.7 - 2.4 ~ O.3 - O.5Jum.The
prevailing ahape of cells iG ~traight, rodlike. Gram-nega-
tive. The colonies growing on pl~in agar are of the convex,
round colourless nsture7 3 - 7 mm in ~ize. On beef-e~tract
broth it forms cloudy gas-evolving sediment. Cultivation
on potato agar re~ult~ in intensive growth with prominent
continuous streak0 and formation of mucu~. Doee not liquefy
gelatin. Curdle~ and aci~i~ies milk.
Aerobe, growa &t 28 - 42C, ceasee to grow at tempera-
tures below 4C.
Relation to car~ohydrate~: as~imilate~ lactose, gluco~e
and maltose with evolution o~ ga~. Doe~ not a~eimilate ars-
binoseO
Relation to hydrocarbon~: a~similates naphthalene,
camphor,octane, hexane, benzene, toluene, xylene~ para~in,
asphaltenee. Para~in oils aa~imilated better than aromatic
type 8 .
Relation to nutrient sources: aaaimilates nitric ni-
trogen.
The strain requires no special nutrients, being capable
of growing on hydrocarbon-depleted media, e.g. stratal wat-
er mineralized to not over 10 g/l, is viable at an ~mbient
temperature from 70C down to minu 50C and its activity
is not aYfected by varyin~ ~eather condition~.
On being dried, the cella retain their viability and
- 8 -
hydrocarbon-oxidizing ability which is superior to
that of the source strain. Observation of -the titer
of a lyophili~ed culture over a 24-month storage
period has conformed its stability.
The claimed strain relates to nonpa-tho-
genic microorganisms. Peroral, intranasal,
intraabdominal and intravenous administration to
white mice, keratoconjunctival and epicutaneous
application to rabbits have revealed its practical
harmlessness and nonpathogenicity. The clinical
observation of the medical staff handling said strain
revealed no allergies nor bacterioses.
According to the invention the disclosed
strain is used in combination with a mixture of
mineral salts. Inasmuch as the strain Pseudomonas
putida-36 is specifically noted for assimilation of
nitrates, nitrate compounds have been given a special
preference in selecting the mineral salts.
It has been found, that the optimum
relation of the main sources of mineral feeds such as
N, P205, K20 is 1:1. Such a relationship can be
obtained with the following composition of mineral
salts, mass %:
KNO334.26 - 37.12
NH4C:L28.66 - 31.28
NH4~2PO425.42 - 28.71
4 32.89 - 11.66
Thus, the mineral component of the claimed
composition delivers 17% N into the habitat of
bacteria (10.2% in the form of NH~Cl, NH4NO3,
NH4H2PO4 and 6.8% in the form
, :: ' - ..
- 9 -
of KN03 and NH4~03); 17% P205 (includin~ 15% of water-~o-
luble P205) in the form of ~H ~2P04 and 17% o~ ~ 0 in the
~orm of KN03.
Said balance of ~, P205 a~d K20 ensures the most
effective utilization of the strain Pseudomona putida-36
for the recovery of water and 80il polluted by oil and oil
products .
The culture of microorgani~ms ma~ be used both in the
native and dry form.
To produce the biomas3 of the cells, the strain Pse~do-
monas putida-36 is grown in liquid or solid nutrient media
containing the source~ of nitroge~, pho~phorus and potassium
in presence of hydrocarbons. Cultivation i~ carried out in
aerobic condition~ at 30. In these condition~ the cell~ of
microorganism~ gro~ and develop vigorously. If need be, the
obtainRd biomass of the cells ca~ be dried by lyophilizi~g
or dry-air spr~ying.
The obtained biomas~ should contain from 1.8 . 101
to 3.6 1012 living cells per 1 g o~ dry mas~.
The composition can be prepared either dry or as an
aqueous aolution.
Preparation of the dry composition, whether with or
without the carrier consists in ~imple mechanical miging
o~ the biocomponent, a mixture of mineral salt~ and, if ne-
ces~ary, a carrier.
~ he cl~imed compo~ition containing a biolo~ical and
a mineral components is a light free-~lowing yellowish powd-
er easily soluble in water9 oil-polluted too.
- 10 -
Preparation of the composition in the form of an aqueou~
solution i~ confined to mixing the biocomponent and ~aid com-
bination of mineral ~alt~ in water.
The dry comp3sition a3 a whole csn be mixed with water;
alternatively, each component, i.eO biocomponent and mineral
component csn be mixed with water sepsrately.
The obtained aqueous compo~ition i6 traneparen-t and
slightly opaque due to the presence of the biological com-
ponent.
The recommended ma~ ratio of the biological component
and mineral salt mixture i~ 1:26 - 32. In other word6,there
are 26 - 32 ma~s part~ of the mineral component (mixture
of salt~) to one part of the biological component. This re-
lationship is goYerned by the fact that the amount of miner-
al component below 26 ma3s parts will be in~ufficient ~or
e~fective activity of bac~eria while its amount exceeding
32 mass parts ~ails to provide additio~al ~timulus to the
vital activity of cells and brings about an unwarranted and
undesirable expenditure of the mineral component.
The term "oil pollution" 3hould be understood as the
presence of crude oil or oil products in a water-dissolYed,
emul~ified and film state in the natural and artificial
water basins, in 6ea water and on area6 of land.
The dry composition ~hould be given preference ~or the
recovery o~ local pollutions of va3t areas or small ~tret-
che~ of damp 30il.
The dry composition i6 readily dis~olved in water 80
that the bacterial cells are quickly and uniformly di~tri-
"~ ~
buted in the water-oil contact zone and are given acce~
to the nutrient sub~trate (hydrocarbons) thus eneuring rapid
growth of the cells and all-round elimination o~ oil pollu-
tions as proved by vi~ual observation~ and control analy~e~.
It is expedient that the wide area~ of both water and
land ~h~ll be recovered ~xo~ oil pollution~ by mean~ of a
composition with a carrier, either liquid, e.g. watar~ or
solid, e.g. talc, diatomite~ paraffin, kaoline and other
inert, light, solid materials. The employment of fil~ers
makes for a more uniform di~tribution of a relat~vely small
amount of the compo~ition over large polluted areas from
airplanes and helicoptersO If a solid carrier is used9 it~
ratio in the claimed composition is 30 - 50 to 1.
The preferable liquid cPrrier i8 a~y kind of water
~ince it i8 generally available, neutral and innoc~ou~ to-
ward~ the object o~ pollution, and becau~e bacteria tend
to grow more intensivel~ in ~ater which proves to b~ moot
esQential when dealing with oil pollutions on dry lands,
sandy and stony areast ~uch ae beaches And ~ea-front~.
Concentrati~n of the biocomponent in an aqueous 80-
lution ranges from 104 to 106 cells/ml. If thi~ concentra-
tion is under 104, it will prove insu~icient ~or the norm-
al growth and development of cells while co~centration above
106 ie unrea~onable since the amount of biocomponent cell~
increa~ee due to their natural multiplication in the pre-
eence o~ hydrocar~ons in oil pollutione.
The above concentration of cells in the aqueous eolu-
tion ie obtained by dissolving 2.5 - 25 g of the biological
~s~
- 12 -
component containing 1~8 . 101 to 3.6 ~ 1012 living cellsin one litre water.
Another object of the invention iB a method of recovery
of oil-polluted water a~d ~oil comprising the application
of said composition to thepolluted areas. The composition
in any form (whether dry or a water ~olution) i~ Qpplied
mostly once to the polluted area where the above-cited pa~
rameters, i.e~ content of biocomponent cells and the amount
of the mineral ~alt mixture make for an active growth of
biocomponent cells and ensure their active vitality. Depend-
ing on the intensity of oil pollution, the amount of appli-
ed compo~ition varies from 350 to 800 mg per ~q.m of the
treated surface. Thi~ amount is sufficient for eliminating
oil pollution of up to 25 kg/m3 of water and up to 10 kg~m -
of soil.
~ he di~clo~ed i~ventio~ is by far more profitable
commercially than the m~thod~ known in the priOr art.
~ he invention proYides a radical sQlution of the environ-
ment protection problem~ particularly the recovery of oil-
polluted water and soil and, what for its technical and eco-
nomical lndices, it outclasses the known methods pur~uing
similar purposes.
An emphatic advantage of the disclosed compo~ition and
the method of its application i~ the increase in the degree
of water and soil recovery to an sb~olute reli~bility aft-
er they have been massiYely polluted with oil and oil pro-
ducts.
The disclosed composition ~implifieL the prQcess of
~2 ~5~
recovery due to its ready-to-u~e form whereas the use of a
pure culture in~olve~ additionsl complications caused bg
the necessity for growiDg the ba~teria on liquid nutrient
media, carrying them to the point of application, additional
control of the concentration of living bacterial cellsl etc.
The disclo~ed composition and the method of application
thereof can be readily realized under industrial conditions
since they do not require any special equipment and special-
ly-trained personnel. ~he method i~ ~ucce~s~ully applicable
in the field.
The disclo~ed composition doe~ not require any parti-
cular storage conditions and it~ application is not a~fected
by weather and climate. The compositlon is noted for stabi-
lity of its properties.
The disclosed composition i3 harmles~ for man and en-
vironment) i-t i8 explosion- and fire-proof.
The discloeed composition can be succes~fully used in
a num~er o~ media, such as aea and ~resh water, induRtrial
sewages, areas of 50il polluted with crude and bunker oil and
the products of itB refining, e.g. die~el fuel, ma~out,lu-
bricants, etc.
The best compo~ition ie the one containing the strain
Yseudomonas putide-36 deposited under No. B-2443 and a mix-
ture of mineral salts, mass %: KN03 - 36.53; ~H4Cl - 30.83;
NH4H2P04 - 27.53 and NX4N03 - 5.11. The content of livi~g
cells in the biological component is about 3.6 ~ 1012 per
gram of dry biomass. ~he ratio of biologicsl to mineral com-
ponents i~ 1:28. The composition is prepared by mi~ing it~
~ 8 ~
- 14
components in the ma~s ratios ~pecified above. The compo~i-
tion i~ a li~ht finely-disper~ed powder, It is practlcable
that said composition be u~ed as an aqueous solution..
This composition iB mo~t effective in comparison with
other formulas since it requires a lower expenditure o~ the
biocomponent and, consequently, permits using the Pame amo-
unt for treating a rnuch larger area of pollution. Thus, in
case of a composition who~e biocomponent contains 1.8 . 101
living cells per g of dry biomass, its consumption rate i8
25 mg per m2 while the biocomponent having 3.6 1012 living
cells per g of dry biomass ca~ be used at the rate of a mere
205 g per m2 which reduces the consumption of the biocompo-
nent 10 times or, in other words, increases the treated area
10 times.
This ver~ion curtail~ considerably the expenditures ~or
tran~portation of the compo~ition.
The present invention will become more apparent from
the appended examples of which e~amples 1 through 3 illustrate
the preparation of biocomponent, examples 4 through 6 des-
cribe the preparation of the dry composition without a car-
rier, examples 7 through 9 describe its preparation in ~n
aqueous solution, example~ 10 to 12 egplain the p~eparation
of the dry compo~ition with a carrier, examples 13 through
illustrate the u~e of the claimed composition for the reco-
very of oil-polluted water a~d soil, and example 18 ~how~
the u~e of a pure strain, mixture of mineral ~alts and of
the di~closed composition
:E~camDle 1
_. .
~his e~ample illustrate~ the preparation of biomass
s~
- 15 -
from the cells of the active component of the compo~itiQn.
The source ~train P~eudomonas putida 36 is grown on a liq~id
nutrient medium in the pre~ence of hydrocarbons, e.g. crude
oil by the deep culti~ation method in a fermenter on the
nutrient medium of the following formula, g:
~P4 10.0
K~2P4 1.0
4N3 2.0
H20 1000.0
Cultivation is carried out under aerobic condition3 at 30C.
The prepared biomass is concentrated and dried by the method
of dry-air spraying at +60C~ The number of living cell~
found by the titration method has been 108 10 per g
of dry matter~
xample 2.
The biomas~ i8 prepared by the technique~ laid down
in Example 1. The obtained bioma~ dried by the dry-air
~praying method at 40C. ~he produced bioproduct contains
2.4 ~ 1011 cell~ per g of dry matter.
Exampl~
The biomass prepared as described in Example 1 i9 dried
by the method of lyophilization. ~he obtained product con-
taine 3.6 1012 living celle per g of dry matter.
ExamPle 4.
1 g of the dry biomass prepared 88 de~cribed in Example
1 is mixed with 25 g mineral salt mixture of the following
composition, maQs ~ 03 - 34.26, ~H4Cl - 28-66, NH4H2P04 -
- 25.42, The prepared composition has the form of a finely
~ - 16 -
~8g~
-dispersed yellowish powder.
Example 5
1 g of the dry biomass prepared as in
Example 2 is mixed with 28 g of a mineral salt
mixture of the following composition, mass%: KN03 -
36.53, NH4Cl - 30.83, NH4H2P04 - 27.53, NH4N03 -
5.11. The produced composition has the form of a
finely-dispersed yellowish powder.
Example 6
To increase the concentration of the
composition, 1 g of the dry composition prepared as
in Example 3, containing 3.6 10 living cells per g
of dry matter is mixed with 32 g of a mineral salt
mixture comprising 37.12% KN03, 31.28% NH4Cl, 28.71%
NH4H2R04 and 2.89% NH4N03. The produced composition
has the form of a finely-dispersed yellowish powder.
Example 7
To make a water solution, 362.5 mg of a
dry composition prepared according to Example 4
should be mixed with 0.5 1 water. The produced
liquid composition contains 10 living cells per ml
and 0.07% of a mineral salt mixture. The prepared
composition is transparent and slightly opaque due to
the presence of the biological component.
Example 8
To prepare an aqueous solution, 540 mg of
the dry composition produced according to Example 5
should be dissolved in 0.75 1 water. The obtained
liquid composition contains 10 cells per ml and
0.07% of the mineral salt mixture. The composition
is transparent and slightly opaque due to the
presence of the biocomponent.
~ 16a -
8S~
Example 9
To prepare an aqueous solution, 825 mg of
the dry composition produced according to Example 6
should be dissolved in 1 1 water. The obtained
aqueous composition contains 10 living bacterial
cells per 1 ml and 0.08% of the
- 17 -
mineral component.
Example 10
~ 0 prepQre a granular composition, 26 g of the dry com-
position containing 1 g of biocomponent and 25 g of a miner-
al ~alt mixture should be mixed with 780 g of paraffin car-
rier. ~he ma~s ratio of the dry compo~ition to the carrier
iB 1: 30. The carrier ~hould first be ground to gran~les
0~05 - 0.1 mm in ~ize and placed into a plate gr~nulator
together with the dry composition. ~he obtai~ed granular
composition i8 recommended for the recovery of oil-polluted
water ~urfaces.
Example 11
To prepare a dry composition, 29 g of the dry compo-
sition produced according to Example 5 and containing 1 g
of biocomponent and 28 g of a mineral ~alt ~ture should be
mixed with 1160 g of kaoline carrier at a ratio o~ 40 parts
of the filler to I masa part of the dry compo~ition. The
mlxture then should be vigorously mixed by mechanical agit-
ators. The obtained compo~ition i8 a light, free-flowing
white powder.
Example 12
To prepare a dry compo~ition, 33 g of the dry composi-
tion produced according to Example 6 and containing 1 g of
biocomponent and 32 g of a mineral ealt mi~ture ~hould be mixed
with 1690~of diat ~ te carrier. The ratio o* dry composition
and carrier is 1:50 mas~ parts. A thoroughly mixed compo-
~ition with the carrier is a light-grey free-flowing light
powder. l'he obtained compo~ition is recommended for the re-
- 18 -
covery of large oil-polluted areas of bogged and water-logg-
ed ~oil~.
E~amPle 13
An area of soil 50 OOO m2 ha~ been polluted by a doae
of 10 ~g of crude oil per m2. In other words, the total
amount of oil in the area wa~ 500 ton~. To eliminate the
oil pollution, 50 m3 o~' the aqueou~ ~olution according to
Example 9 wa~ u~ed. Each litre of the compo~ition contains
825 g of a mixture of biocomponent with mineral ~alt~. The
composition i8 spread uniformly at the rate of 1 l/m by
spraying from ~elf-propelled ~prayers equipped with pre~sure
pumps capable of delivering the pulverized jet o* liquld
to a di~tance of 35 - 45 m. On expiration o~ 2 months after
a ~ingle treatment, the amount of destroyed oil was 460 t.
The experiment was conducted in sunny weather alte~nating
with rain ~ the temperature varyi~g from ~26 to 0~C. Ob~er-
vation~ revealed active plant vegetation in the polluted
area.
Example 14
A 9ection of a natural fre~h-water bàsin within the
territory of an oil field has been polluted with crude oil
over an area of 25 OOO m2 at the rate of 23.3 l per m3 which
mean~ that the total amount of crude oil in the section was
581.5 t~ To eliminate oil pollution a water composition pro-
duced according to Example 8 was u~ed. Said composition
contained 543.75 mg of dry composition in which 18.75 mg of
biocomponent and 525~g of a mineral salt mixture were pre-
~ent at a ratio of 1:28 and the water solution contained
s~
-- 19 --
105 li~ing bacterial cell per ml a~d 0.07% of the mineral
salt mixture- The CompOBitiOn Was prepared in the amount of
18.75 m3 at the rate of 0.75 1 per 1 m of the polluted BUr-
face. The water composition was applied once by uniform aeri-
al spraying. On expiration of 2 months after treatment the
basin wae completely recovered from oil.
Example 15
An object of treatment is open ~edimentation basin~
for industrial s0wa~e of ~n oil refinery. The total area of
the basin is 800 000 m2, the volume of water, 1.5 million
m3. The content o~ oil products (v~ater-~oluble, emulsified
and in a ~ilm state) amount~ to 75 mg/l.
~ he oil pollution i~ eliminated b~ the use of a compo-
~ition proùuce~ accordi~g to Example 7 and containing 2.5 kg
of biocomponent and 70 kg of a mineral salt mixture dis~olv-
ed in 400 m3 o~ water; this composition cont~ins 104 living
bacterial cells a~d 0007% of the mineral ~alt mixture per ml
water.
The composition i8 di~tributed uniformly over the en-
tire aree of the water basin at the rate of O.5 1/m2 from
a fire truck equipped with a tank and a carriage-mounted fire
hose.
14 day~ after a single treatment the content of oil
producta in water has dropped to 0.5 mg/l, remaining stable
at this level within the following 14 day~ in ~pite of a
con~tant inflow of the indu~trial sewage laden with 75 mg/l
of oil product~.
~ s~
20 -
Example 16
A glas~ reservoir (1.25 m long, 0.8 m wide, 0.15 m
high) is filled with 100 l of watex ~rom the Kara Sea and
1720 g of crude oil.The oil film i8 removed by u~ing 22.5 g
of the composition prepared in accorda~ce with E~amplè 10.
This composition contain~ 25 mg of biological component,
700 mg of mineral component and 21.75 g o~ paraffin carrier.
At a room temperature of 18C the sea water wa~ completely
puri~ied of oil within 14 days after a single treatment.
Example 17
A 200 m boggy area of ~oil polluted with 60 kg o~ oil
(0~3 kg/m ) was treated ~ith 7 500 g of the dry compo ition
prepared according to Example 12. The dry composition wa~
uniformly sp ayed oYer the polluted area at the rate o~
37.5 g per m . This dose contained 25 mg of the biocompo~
nent, 750 mg of a mineral salt mixture and 36,75 g of the
inert diatomite carrier. 22 day~ after a single treatme~t
the area wa~ fully cleaned from oil.
E~ample 18
~ he given example illustrates the employment of a pure
strain Pseudomona~ putida 36, or of a mineral salt mix-ture
or of their combination9 i.e. the claimed composition for
elimlnation of oil pollutiona. The results gained are ~um-
marized in the Table below.
It can be seen from the Table that the pure strain
P~eudomonas putida-36 decomposes 68-76% of the initial amo-
unt of oil; a mixture of mineral salt~ (w/o strain), 6.4
- 8.13~; and the claimed composition, 98.9 - 99.9~.
_ 21 -
Table
Re~ults of rTe Bt8 of Strain Pseudomonas Putida-36, I~ture of
Mineral Salts and Claimed Compo~ition for RecoYery o~ Oil-
Polluted Water ~nd ~oil
Condition~ Oil de~radation ef~i-
of Treating material Sample _ cienoy, %
experiment ~0 aromatic paraflin
Oil-pollut- Strain P~eudomo- 1 69~1 72~8
et water~ nas putida-36 2 87 ~ 5 76 ~ 7
l~o YOl. 3 6~3o72 7309
Duration of 68. 44 74 ~ 46
experiment ~ineral ~alt mix~ 1 6D7 ' 7~1
96 - 120 h. ture, %:
~KNO3 36053 2 6.9 5.8
~I4Cl 30.83 3 5.6 6.7
N~4E2P04 27 ~ 53 6.4* 6.53
4 3 5~ 1
Claimed compo~ition
containing strain 1 98.6 99.8
Pseudomona~ putida- 2 98.9 100.0
36 and mixture o~ 3 99.7 * 10090 *
aaid mineral ~alta 99.06 99.93
Oil-polluted Strain Paeudomonaa 1 73 . 9 75
~oil,10% vol putida-36 . 2 68.2 74 9
3 72.7 79.2
Duration of 71 ~ 6 76.4
experiment
20 - 30 daya Mineral ~alt mix- 1 7.7 7.9
ture, % 2 7.9 8.0
3 6.9 8.5
7.5 8.13
KN03 36.53
NH4Cl 30~83
H2P04 27~53
4 3 5.11
Claimed composition 1 98.4 99.8
containing atrain 2 98.7 10000
Pssudomonas putida- 3 99.6 99.9
36 and mixture of ~ *
~aid mi~eral ~alts 98-9 99-9
, ..... . , . . , . ,~
* mean value
- 22 -
The u~e of the mineral salt mixture in combin~tion with
the pure ~train P~eudomonas putida-36 raises the efficiency
of sQid ~train 1~31 - 1.45 time~ on the average and i~ cap-
able of ensuring practically 100% recovery of the media pol-
luted with oil and oil product~.
