Process of culturing microorganisms using a microemulsion

There is described a process for the culture of microorganisms in a medium comprising a hydrophobic material containing microorganisms by the use of nutrient substances in aqueous solution micro-emulsified in a liquid immiscible with water. As the source of nitrogen, compounds soluble in water are used, such as urea or ammonium nitrate, sulphate or phosphate, and, as the source of phosphorus, compounds soluble in water are used. The medium should also contain a directly-assimilable source of carbon. Application of this process to the culture of microorganisms, allowing operations to be carried out which comprise the degrading of hydrocarbons covering an area of water or ground; an application of particular interest for operations for combatting oil pollution of the sea by biological degradation.

The present invention relates to a novel type of microemulsion, namely the 
thermodynamically-stable microemulsion of an aqueous solution of nutrient 
materials, the external phase of which is constituted by a hydrophobic 
medium. It comprises a process of production of such a microemulsion and 
applications of it in areas of utilization of microorganisms, particularly 
in the culture of microorganisms in a hydrophobic medium. 
Industrial operations making use of cultures of various microorganisms, 
particularly bacteria and fungi, are of particular interest at the present 
time. Numerous food industries, manufactures of medicaments, purifications 
etc. are based upon such operations. Most frequently, the operation 
comprises two stages: Firstly, culture of the microorganism concerned 
takes place in an appropriate nutrient medium to achieve growth of a 
sufficiently numerous population; in the second stage, this population is 
placed in contact with materials which it is desired to subject to the 
action of the microorganisms. Operations in vessels do not involve special 
difficulties from the standpoint of, preliminary culture, that is, 
multiplication, being effected--depending upon the case--in a separate 
enclosure or in the same vessel where the second operative phase takes 
place. In contrast, when the operations are carried out in nature, on 
large areas of ground or water, as is the case for example in the 
elimination of hydrocarbon slicks by microbiological degradation at sea, 
on beaches, in flowing water or on lakes, the preliminary multiplication 
of the microorganisms used presents difficulties. In practice, it is 
necessary to provide the culture with nutrient materials, that is sources 
of C, N and P, as well as trace elements, to ensure growth of the 
organisms in question. When the standard sources, such as hydrides of 
carbon, nitrates or ammonical salts and phosphates are soluble in water, 
they cannot remain in the extended superficial layer to be treated, where 
a strong growth of the microorganisms used should occur. These substances 
diffuse into the water or the adjacent ground and are thus under-used by 
the culture. 
To remedy this, various methods have been employed up to the present. One 
of them consists in enrobing in paraffin granules of the nitrogenous 
compound and solid phosphates in order to make them available to the 
microorganisms in this form, as indicated in U.S. Pat. No. 1,959,127. In a 
variant, according to the U.S. Pat. No. 3,883,397, the lipophilic enrobing 
compound is a fatty acid salt in place of paraffin. However, these methods 
do not allow the microorganisms to have the desired nutrient rapidly 
available. The fatty enrobing material is difficult to penetrate, that is, 
to degrade, in the absence of external nitrogen and phosphorus. Also, the 
action of bacteria or fungi itself is slow and requires weeks or months. 
Another proposed solution consists in utilizing, as the sources of N and 
P, compounds insoluble in water but soluble in hydrocarbons, particularly 
phospho-amino-lipids, as described in French Published Specification No. 
2172796. However, nitrogenous compounds soluble in oil generally have a 
very low nitrogen content and, under these circumstances, the biological 
degradation of petroleum hydrocarbons in the sea requires two to three 
months. In French Published Specification No. 2230401, the use has also 
been indicated of amides, organic ammonium salts and phospho-amino-lipids 
in solution in a petroleum solvent, the solution being emulsified in 
water. The emulsion obtained is atomized over a hydrocarbon oil slick 
floating on the water for biodegradation of the slick. This method 
requires large proportions of the aqueous emulsion and the result is only 
obtained after several weeks. 
The present invention provides a novel solution to the supply of nutrient 
substances, soluble in water, to a hydrophobic organic layer. Whether this 
layer--which can be a hydrocarbon--floats on water or is disposed on the 
ground or on a support comprising a constructional material, the nutrient 
substances provided according to the invention remain principally in the 
hydrophobic layer and permit rapid multiplication of the microorganisms if 
sources of them are present. 
The novel process according to the invention consists in making a 
microemulsion of the water-in-oil type, the internal phase of which is an 
aqueous solution of nutrient materials and the external phase of which is 
a liquid immiscible with water, and adding this microemulsion to the 
hydrophobic layer which is to be degraded. The microemulsion can contain 
sources of the appropriate microorganisms, if the medium to be treated 
contains none or does not contain sufficient amounts. 
It will be understood, as is known in the art, that the microemulsion 
contains at least one surface-active agent and an auxiliary agent, which 
have served in its preparation. 
Thus, contrary to the prior technique, the nutrient substances are neither 
utilized in the solid state nor in solution in a solvent immiscible with 
water nor in an aqueous macroemulsion, but in the form of a 
microemulsified aqueous solution in a liquid miscible with the hydrophobic 
layer to be biodegraded, that is, in the form of microdispersed so-called 
inverse droplets, the diameter of which ranges from 80 to 600 Angstroms 
and in particular from 100 to 200 Angstroms. This unexpected form leads to 
the remarkable result that the biodegradation can be realized in several 
days, in place of the weeks or months required by known processes. 
As regards the source of nitrogen, in the microemulsions according to the 
invention, various compounds soluble in water and assimilable by the 
microorganisms can be employed. These are, for example, ammonium nitrate, 
sulphate and/or phosphate, urea, proteins, peptones etc. As urea is the 
source richest in nitrogen and is very soluble in water, it is 
particularly appropriate because it allows highly concentrated aqueous 
solutions to be produced. For example, solutions of urea containing 10% to 
60% by weight can be employed, that is, 11 to 150 parts of urea per 100 
parts of water. 
Phosphorus can also be provided in the solution in one of the customary 
forms, namely alkali metal or ammonium phosphates or phosphites. According 
to a particular embodiment of the invention, phosphorus is provided in the 
form of a surfactant compound, such as a higher alkyl phosphate or a 
lecithin, for example. The source of phosphorus and the surface-active 
agent of the microemulsion are thus in the same molecule. 
In various industrial operations comprising the culture of microorganisms, 
it is necessary for the pH of the medium to be adjusted to the value most 
favourable to bacterial growth. In general, this pH should be in the 
vicinity of neutrality and it is possible to add either phosphoric acid as 
a source of phosphorus, if the medium has to be acidified, or ammonia as a 
source of nitrogen, if the medium has to be neutralized by a base. 
In the case of bacterial degradation of hydrocarbons by the microorganisms 
cited below, the need for phosphorus is much less than for nitrogen; 
expressed in weight, the P/N ratio can range from 0.02 to 0.2 and 
preferably from 0.05 to 0.15. From the standpoint of growth, the most 
favourable P/N ratios are those as near as possible to 0.05. 
When the liquid immiscible with water and preferably lipomiscible, forming 
the external phase of the microemulsion, or the hydrophobic layer to be 
degraded is utilizable by the microorganisms as the source of carbon, it 
is no longer necessary to add other assimilable carbon compounds to the 
microemulsion. However, if the external phase and the layer to be degraded 
are attached with difficulty by the microorganisms, at least at the 
beginning, it is useful to include in the nutrient solution a 
readily-utilizable carbon source, for instance soluble hydrides of carbon, 
thus allowing rapid onset of multiplication of the microorganisms. 
As in all cultures, trace elements are necessary, particularly salts of Fe, 
Mg, K etc., and a very small dose can be added to the nutrient solution, 
in known manner. 
It can be seen that, in order to obtain a microemulsion according to the 
invention, a surface-active compound capable of producing one is 
necessary. The choice of a suitable compound, by a person skilled in the 
art, can be made from various groups of non-toxic surface-active agents 
for the microorganisms present. For example, fatty alcohol sulphates, 
sulphosuccinates, oxyethylenic sorbitan esters, oxyethylenic alcohols, 
acids or oils, esters of saccharose, amino-acids, alpha-amido-amino acids, 
taurines, sarcosines, polyglycols, higher alkyl phosphates etc. can be 
used. This list is not limitative and other surfactants can be utilized, 
in particular those which have dispersant properties vis-a-vis 
hydrocarbons. 
Preferably, the hydrophilic-lipophilic balance of the emulsifying agents 
employed is from 10 to 17 and most preferably from 11 to 15. 
When operations are concerned which are carried out outside, the surfactant 
itself must be biodegradable, in order to avoid environmental pollution. 
As with the surfactants, the choice is equally vast as regards the 
co-surfactant necessary for the formation of the microemulsion. Such 
co-agents are well-known in the art and thus they do not need to be listed 
here. It can merely be noted, in a non-limitative way, that it is possible 
to use nitrogenous compounds, such as carbamates, amides or amine salts. 
The viscosity of the microemulsion can be considerably reduced by the 
addition of an alcohol, particularly C.sub.6 to C.sub.12, an ether or an 
ester of a polyol, particularly glycol. This considerably facilitates 
manipulative operations. 
When the external phase of the microemulsion must be miscible with the 
hydrophobic liquid to be biodegraded, it is necessarily chosen according 
to the nature of this liquid. In the most important practical case, where 
the latter is constituted by petroleum hydrocarbons, the lipomiscible 
external phase can be constituted for example by aliphatic, aromatic or 
naphthenic hydrocarbons or by so-called mineral oils, that is mixtures of 
such hydrocarbons. This type of external phase is attached with difficulty 
by bacteria, when these have not undergone sufficient adaptation. It is 
thus preferable to utilize vegetable or animal oils, which can serve as 
the source of carbon because they are utilizable by microorganisms. These 
oils or preferably their corresponding fatty acids permit rapid 
development of the microorganisms necessary for the degradation of the 
hydrophobic layer, particularly crude petroleum. 
The weight ratio between the lipomiscible liquid, that is the external 
phase of the microemulsion, and the aqueous solution to be emulsified 
should generally be greater than 0.2. This ratio is selected so that the 
aqueous solution forms the internal phase. The choice of surfactants and 
co-surfactants is effected according to the nature of the lipomiscible 
liquid and depending upon the concentration of the salts dissolved in the 
aqueous phase. The basis for this are the concepts of formulation of 
microorganisms, known per se. 
The process of the invention is applicable to a large number of 
microorganisms and in particular to those which allow the degradation of 
hydrocarbons. Thus, the invention can be applied to the utilization of 
bacteria such as Pseudomonas, Acinetobacter, Flavobacterium, Artrobacter, 
Corynebacterium etc. The microorganisms can also be fungi. 
While the invention is of great interest for various operations of 
biodegradation effected outdoors, it can also be of use in various 
manufacturing operations in vessels, whenever a hydrophobic layer of a 
substance is employed in the process. For example, it applies 
advantageously in the manufacture of proteins from hydrocarbons by 
degradation of the latter with the aid of bacteria and/or fungi. In all 
cases, the remarkable dispersion of aqueous nutrient substances within the 
hydrophobic phase, obtained through the invention, leads to a very rapid 
multiplication of the microorganisms. An appreciable gain in time in these 
operations thus results. 
Among applications in the open, water or ground areas, the most important 
is the degradation of hydrocarbons distributed accidentally. For the 
reason explained above, that is the fact that the soluble nutrient 
substances remain in the layer treated instead of being entrained by the 
water, the invention has a considerable value for combatting marine 
pollution. However, the same principle applies to operations such as the 
cleansing from banks, reservoirs, ground areas, containers etc. of 
hydrocarbon deposits which pollute them. Other applications comprise the 
distribution of nutrients on agricultural cultures. 
Microorganisms are generally present in the medium being treated. However, 
it is sometimes necessary to effect seeding, when the initial population 
is judged to be too low or if the medium contains no appropriate bacteria. 
In a particular embodiment of the present invention, urea is utilized as 
the nitrogenous nutrient substance. It has been found that this compound 
also plays the part of the co-surfactant and it is thus no longer 
necessary to add another co-surfactant. On the other hand, as phosphorus 
can advantageously be provided by alkyl esters of phosphoric acid, which 
provide surfactant properties, the composition of the nutrient solution is 
simplified by the fact that it is possible to employ urea and the 
phosphoric ester without any other additive. It is nevertheless 
recommendable to add liquids permitting reduction of the viscosity of the 
microemulsion. Various examples of such additives have been cited above. 
In a particular embodiment of the invention, the butyl ether of ethylene 
glycol has given excellent results. 
The lipomiscible liquid particularly suitable for the external phase of the 
microemulsion, according to the invention, can be constituted by one or 
more esters of fatty acids, such as lauric, myristic, palmitic, arachidic, 
oleic, stearic, caproic and caprylic acids etc. Glycerides of such acids 
constitute very readily available industrial products, because they are 
vegetable and animal oils. Thus, oils such as arachidic, whale, colza, 
linseed, maize, olive, sesame and tall oils etc., can be used. Fatty acids 
themselves are particularly suitable, if required in appropriate mixtures, 
in order to remain liquid at ambient temperatures. Thus fatty acids are 
useful, particularly those containing C.sub.6 to C.sub.18, such as 
caproic, oenanthylic, caprylic, lauric, palmitic, oleic, lineoleic or 
stearic for example. To those fatty materials which are not liquids at 
ordinary temperatures, it is suitable to add hydrocarbons, for example 
petroleum or gas oil in the proportion of about 5% to 50%. Fatty alcohols, 
that is from C.sub.6 to C.sub.24, are equally suitable. 
In a particular case, where the aqueous solution contains urea and lauryl 
and/or oleyl phosphates marketed for example by Hoechst under the name 
"Hostaphat", the preferred nitrogen content of the entire microemulsion is 
about 4% to 10% by weight or most preferably 5% to 8%. The weight ratio of 
nitrogen in the lipomiscible liquid is generally from 0.1 to 0.4 and 
preferably 0.15 to 0.35. 
In a general manner, the preferred microemulsions according to the 
invention comprise by weight 10% to 30% of water, 4% to 10% to assimilable 
nitrogen in the form of nitrogenous compounds, 5% to 35% of a C.sub.10 to 
C.sub.18 alkyl phosphate or an ethoxylated phenol alkyl phosphate, 0% to 
20% of an alkyl ether of an alkylene glycol and 20% to 50% of an aliphatic 
ester, acid and/or alcohol. The compounds can comprise liquid 
hydrocarbons, such as petroleum or its derivatives, for example in the 
ratio of 5% to 70%. 
A variant of the invention comprises an improvement which allows a very 
rapid action of the microorganisms to be obtained. This renders possible 
the degradation of hydrocarbons in a very short time, by utilizing for 
this purpose a very large number of microbes. It has been discovered that, 
while the best nutrient substance is urea, a part of the microorganisms 
normally present in seawater and capable of degrading hydrocarbons do not 
develop and consequently do not participate in the desired degradation. 
According to the present variant, this fraction of the microorganisms 
which remain "inactive" can be caused to develop and participate in the 
degradation of the hydrocarbons, if the nitrogenous nutrient material is 
accompanied by one or more nitrogenous materials of a chemical composition 
substantially different from the first. Particularly favourable results 
are obtained when the first nutrient material is urea and the second is 
constituted by one or more amino-acids. 
It follows that a microemulsion according to the invention intended for the 
microbiological treatment of a hydrocarbon material preferably contains an 
aqueous solution of at least two nitrogenous compounds which are 
substantially different from the chemical standpoint. Thus, for example, 
if the first nutrient substance is a salt such as ammonium sulphate, 
phosphate or nitrate, the second is constituted by an amine, an amide, a 
protein, an amino-acid or another non-ammoniacal compound. 
When the nutrient solution comprises urea, the second nitrogenous compound 
is, for example, ammonium sulphate, phosphate or nitrate or an amino-lipid 
and particularly an amino-acid. The relative proportions of the two kinds 
of nitrogenous materials can vary largely, depending upon the nature of 
the microbial flora of the medium where the process of the invention is 
applied. Most frequently, the effective proportion of urea, expressed as 
nitrogen, is from 50% to 99% of the total nitrogen, that is the nitrogen 
of the amino-acid represents 50% to 1%. In certain aqueous media, about 1% 
to 10% of nitrogen can be sufficient in this second form, in order to 
obtain excellent results. 
The amino-acids advantageously utilizable according to the present 
invention can be selected from all those which are found in nature and 
from synthetic amino-acids. By way of non-limitative examples, use can be 
made of glycine, alanine, serine, cysteine, valine, glutamine, leucine, 
lysine, arginine, proline, tyrosine, aspartic and glutamic acids etc. For 
reasons of economy, it is useful to utilize the materials obtained from 
natural products which generally contain a series of several amino-acids. 
This is the case for example with wines made from sugar beet, extracts 
obtained by the maceration of various plants, particularly maize cobs, 
yeast extracts, products of the hydrolysis of proteins, dairy by-products 
etc. 
The invention leads to the unexpected conclusion that, if a nitrogenous 
nutrient material gives good results alone and if the same applies to 
another nitrogenous material of a different chemical nature when used 
alone, the degradation of hydrocarbons by microorganisms is much better, 
if the two materials are utilized conjointly, the total concentration of 
assimilable nitrogen being the same. 
Thus, microemulsions according to the invention allow a degradation of more 
than 80% of crude petroleum distributed on seawater to be obtained, for 
example, in seven days, when the nutrient solution contains urea or 
amino-acids. But the same result is obtained in six days, if the urea and 
the amino-acids are present conjointly in the solution, the total nitrogen 
concentration thereof being the same as in the two preceding cases.

The invention is illustrated by the series of non-limitative examples which 
follow. 
Examples 1 to 11 
For each of the tests, a certain volume of a 50% by weight aqueous urea 
solution was mixed with a volume of oleic acid in the presence of a 
certain quantity of surfactants constituted by a mixture of C.sub.12 to 
C.sub.18 fatty alcohol phosphoric esters marketed by Hoechst Company under 
the name "Hostaphat". In certain of the tests, the butyl ether of ethylene 
glycol was also added to lower the viscosity. The range of temperatures in 
which the microemulsion obtained was stable was determined. 
Table 1 on the following page indicates the compositions of the 
microemulsions so prepared, the stability ranges of the latter and their 
viscosity. It can be seen that an excellent stability in the range from 
0.degree. C. to more than 40.degree. C. can be obtained according to 
Examples 4, 5, 6, 8, 10 and 11. As regards viscosity, it was found that, 
without the addition of the butyl ether of ethylene glycol, it is very 
high (Examples 1 and 2). In contrast, this addition reduces it to very 
acceptable values (Examples 3 to 11). 
TABLE 1 
__________________________________________________________________________ 
1 2 3 4 5 6 7 8 9 10 11 
__________________________________________________________________________ 
Water 11.1 11.7 13.8 
14.4 
18.3 
20.1 
20.9 
21.9 
19.2 
20.2 
19.2 
Urea 11.1 11.7 13.8 
14.4 
18.3 
20.1 
20.9 
17.2 
15.8 
16.6 
15.7 
Oleic Acid 44.4 46.7 36.8 
38.4 
36.6 
32.1 
27.9 
27.9 
28 29.4 
28 
Butyl ether of ethylene glycol 
0 0 10.6 
9.8 10.7 
11.6 
14.4 
14.4 
17.5 
16.2 
10.5 
Lauryl Phosphate (KL 340) 
33.3 -- 25 -- -- -- -- -- 19.5 
8.8 
19.5 
Oleyl Phosphate (KO 380) 
-- 30 -- 23 -- -- -- -- -- -- -- 
Ethoxylated Phenol Alkyl Phosphate 
-- -- -- -- 16.1 
16.1 
15.9 
15.9 
-- 8.8 
-- 
Mono Ammonium Phosphate 
-- -- -- -- -- -- -- 2 -- -- -- 
% Nitrogen 5.2 5.45 6.44 
6.72 
-- -- -- -- 7.4 -- -- 
Nitrogen/Oleic acid ratio 
0.117 
0.117 
0.175 
0.175 
0.23 
0.29 
0.35 
0.31 
0.26 
0.26 
0.26 
Phosphorus/Nitrogen ratio 
0.154 
0.064 
0.09 
0.043 
0.099 
0.09 
0.086 
0.063 
0.064 
-- -- 
Stability Range .degree.C. 
10-44 
10-46 
1-44 
0-44 
0-40 
0-47 
12-57 
0-40 
0-36 
0-42 
0-45 
Viscosity in cps at 20.degree. C. 
1200 2000 167 265 203 197 141 98 61 -- -- 
__________________________________________________________________________ 
Examples 12 to 16 
Microemulsions were prepared with 33% lauryl phosphate. The lipomiscible 
liquid was oleic acid in a quantity equal to twice that of the aqueous 
solution of urea and phosphate. The percentage of urea in the aqueous 
phase was varied. The results below give the maximum temperature at which 
the microemulsion was still stable. 
______________________________________ 
% Urea in the aqueous 
Upper limit of the 
phase temperature .degree.C. 
______________________________________ 
22.2 36 
25 32 
33 38 
50 35 
56 No microemulsion 
______________________________________ 
The results shows that microemulsions viable in practice, stable up to 
about 36.degree. C. can be obtained with concentrations of urea ranging up 
to 50%, but not above that. 
Examples 17 to 19 
With a 50% aqueous solution of urea and the addition of oleyl phosphate 
containing 30% of the mono-ethyl-ether of ethylene glycol, three 
microemulsions were prepared containing varying proportions of oleic acid. 
The stability ranges of these products were as follows: 
______________________________________ 
Weight ratio of the Stability 
urea oleic acid Surfactant range 
solutions (Oleyl phosphate) 
.degree.C. 
______________________________________ 
0.5 25.3 0-65 
0.625 24.2 0-59 
0.75 26.7 O(gel)-64 
______________________________________ 
This shows that above an aqueous solution/oleic acid ratio of about 0.65 
and for a fixed quantity of the mono-ethyl-ether of ethylene glycol of 
30%, the microemulsion is difficult to utilize because it tends to gel. In 
contrast, below this ratio, excellent stability is confirmed. 
DEGRADATION OF PETROLEUM HYDROCARBONS 
Example 20 
30 l of seawater sterilized at 120.degree. C. for 2 hours was introduced 
into a 50-liter fermenter. On the surface of the seawater, 30 ml of 
34.degree. API crude petroleum containing 75% saturated hydrocarbons and 
25% aromatics was spread out. The petroleum layer had a thickness of 0.5 
mm. On this layer, 6 ml of one of the microemulsions of the foregoing 
Examples was atomized. The medium was thus seeded with sources derived 
from the seawater. These sources were obtained by culturing for 24 hours 
on an aqueous solution of glucose. They contained Pseudomonas in a major 
part. 
After this inoculation, the sources in the petroleum samples were assayed; 
2.5.times.10.sup.3 to 4.times.10.sup.4 microbes per ml were thus found. 
Aerobic culture was then carried out by agitating the contents of the 
fermenter with an agitator rotating at 400 revs per min, while 120 liters 
of sterilized air per hour were blown in. This aeration corresponds 
substantially to that which takes place naturally at sea. 
After 40 hours, a further assay was effected. The results are indicated 
below. 
After seven days, the rate of degradation of the petroleum was determined 
by extraction of the residual hydrocarbons with CCl.sub.4 and infrared 
measurement. The results obtained with the microemulsions of Examples 1, 
2, 3, 10 and 11 are indicated below. 
______________________________________ 
Microemulsion Rate of degradation 
of Initial Source assay 
after 
Ex. source after 48 hours 
7 days 
______________________________________ 
1 2.5 .times. 10.sup.3 
2.5 .times. 10.sup.8 
83% 
2 2 .times. 10.sup.4 
9.5 .times. 10.sup.8 
90% 
3 1.5 .times. 10.sup.4 
2.5 .times. 10.sup.8 
90% 
10 4.5 .times. 10.sup.3 
2.5 .times. 10.sup.8 
86% 
11 1.5 .times. 10.sup.3 
7.5 .times. 10.sup.7 
92% 
______________________________________ 
Example 21 
A test was carried out according to the mode of operation described in 
Example 20, but non-sterilized seawater was introduced into the fermenter 
and the medium was not seeded with supplementary bacteria. 
The results are: 
______________________________________ 
Microemulsion Rate of degradation 
of Initial Source assay 
after 
No source after 40 hours 
7 days 
______________________________________ 
11 2 .times. 10.sup.3 
5.5 .times. 10.sup.7 
82% 
______________________________________ 
Example 22 
A test was carried out according to the mode of operation of Example 20, 
but the seawater was replaced by natural water into which various mineral 
constituents of seawater had been added, namely 30 ppm of trace elements, 
particularly iron, magnesium and potassium. 6 ml of microemulsion No. 11 
was atomized and the medium was seeded with a culture of bacteria. 
After 7 days, the rate of degradation of the petroleum was determined by 
extraction of the residual hydrocarbons with CCl.sub.4 and infrared 
measurement; the rate was 90%. 
Examples 23 to 24 
Biodegradation tests on crude Arabian petroleum were effected at the edge 
of the sea in a vessel having a depth of 2 meters, divided into four 
compartments each 3 m.times.3 m in horizontal section. The compartments 
could be isolated or connected together and all were capable of receiving 
seawater. 
A hydro-ejector pump system ensured light agitation of the water and 
renewal of the air in the vessel. 
Into each compartment, 15.6 m.sup.3 of seawater and 4 liters of the 
petroleum indicated above were introduced, thus providing a layer 0.45 mm 
thick on the surface of the water. 
One of the compartments served as a control; the water and the petroleum 
contained in it were agitated as in the other compartments, but received 
no additive. At the end of the test, the losses of petroleum due to 
natural causes were determined in order to take them into account in 
evaluation of the degradation caused by the additions according to the 
invention. 
In each of the three other compartments, 0.4 l of a microemulsion of a 
nutrient solution was introduced, comprising in % by weight: 
______________________________________ 
urea 17.0 
clear water 20.8 
butyl glycol 10.8 
lauryl phosphate 21.1 
oleic acid 30.3 
______________________________________ 
At the start of the test, the microflora of the seawater in the tank 
comprised 10.sup.2 bacteria per ml. After 7 days the quantities of 
petroleum which had disappeared from the compartments were determined. The 
Table below indicates the amounts in % of the initial quantity at two 
different temperatures. 
______________________________________ 
Con- Example Con- Example 
trol 23 trol 24 
______________________________________ 
% of disappearance due 
to natural causes 
5 5 17.5 17.5 
% due to treatment 
according to the 
invention 0 58 0 61.3 
% total disappearance 
5 63 17.5 78.8 
______________________________________ 
As can be seen, even at the relatively low temperature of 12.degree. C., 
the biodegradation obtained in seven days by the treatment of the 
invention is remarkable. In contrast to most of the known processes 
without the use of a culture of microorganisms, this was obtained with the 
sole use of those existing in the seawater. 
Example 25 
30 ml of seawater were introduced into a 50-liter fermenter. On the surface 
of this water, there was spread 30 ml of crude 34.degree. API petroleum, 
containing 75% of saturated hydrocarbons and 25% aromatics. On the 
petroleum layer thus formed, having a thickness of 0.5 mm, 6 ml of the 
microemulsion having the following weight composition was atomised: 
______________________________________ 
urea 17.3% 
water 21.5 
butyl ether of ethylene glycol 
10.8 
lauryl phosphate 23.7 
oleic acid 26.7 
(8.07% of nitrogen) 100.0 
______________________________________ 
Assay of the bacteria in the petroleum indicated the presence of 10.sup.2 
bacteria per ml. 
Aerobic culture was then carried out by agitating the contents of the 
fermenter with an agitator rotating at 400 revs per min while 120 liters 
of sterilized air per hour were blown in. This aeration corresponded 
substantially to that which takes place naturally at sea. 
After 48 hours, a new assay was effected. It showed the presence of 
2.5.times.10.sup.8 bacteria per ml. 
After 7 days, the rate of degradation of the petroleum was determined by 
extraction of the residual hydrocarbons with CCl.sub.4 and infrared 
measurement. This rate was 83%. 
Example 26 
Identical operations to those of Example 25 were effected, but in the 
microemulsion a part of the urea was replaced by the amino-acid DL-valine. 
The weight composition of the microemulsion was: 
______________________________________ 
urea 16.8% 
DL-valine 2.0 
water 20.5 
butyl ether of ethyleneglycol 
10.8 
lauryl phosphate 23.7 
oleic acid 26.2 
(total nitrogen, of 100.0 
the urea and valine, 8.07%) 
______________________________________ 
Bacterial assay gave the number as 10.sup.2 at the start and 10.sup.9 after 
48 hours. The rate of degradation of the petroleum was 84% by the sixth 
day. 
Comparison with Example 25 shows that addition of the amino-acid allowed 
10.sup.9 bacteria to be obtained in place of 2.5.times.10.sup.8 with urea 
alone after 38 hours. The degradation was of the same order (84% against 
83%), but was obtained more rapidly, in six days, whilst it was necessary 
to provide 7 days in the case of urea alone. 
Example 27 
According to the technique of Examples 25 and 26 a microemulsion was 
utilized in which a part of the urea was replaced by an aqueous extract of 
maize cobs containing a series of amino-acids, predominantly alanine, 
arginine, glutamic acid and leucine. Other amino-acids present in lesser 
proportions were proline, isoleucine, threonine, valine, phenyl-alanine, 
methionine and cystine. The total nitrogen content in this extract was 1%. 
The microemulsion had the weight composition: 
______________________________________ 
urea 12.4% 
corn-cob extract 18.7 
butyl ether of ethyleneglycol 
19.2 
lauryl phosphate 29.1 
oleic acid 20.6 
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The water of the aqueous phase was that of the aqueous extract. The total 
nitrogen content of the aqueous phase amounted to 6%. 
Starting at 10.sup.2 bacteria, they numbered 4.times.10.sup.9 after 48 
hours and the rate of degradation of the petroleum had attained 88% after 
six days. 
Comparison of these results with those of Example 25 shows the desirability 
of the addition of amino-acids to urea. 
Examples 28 to 37 
In this series of tests analogous to Example 27, the lauryl phosphate was 
replaced by oleyl phosphate and oleic acid by various liquids indicated in 
the following table, which gives the rate of degradation of petroleum 
obtained after 6 days. 
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Example No 
Hydrophobic liquid utilized 
Degradation % 
______________________________________ 
28 Arachidic oil 88 
29 Colza oil 82 
30 Tall oil 86 
31 Mixture of copra fatty acids 
with 10% oil of vaseline 
85 
32 Lauric acid liquefied with 
10% of crude petroleum 
84 
33 Butyl caproate 85 
34 Ethyl laurate 87 
35 Methyl oleate 86 
36 Amyl stearate 88 
37 Gas oil with 10% sesame oil 
83 
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Similar results are obtained with a mixture of C.sub.12 -C.sub.14 alkyl 
mono-, di- and tri(alkyltetraglycolether)-o-phosphates, known commercially 
under the name "HOSTAPHAT KL 340 N", instead of oleyl phosphate.