FR2695139A1 - Biodegradation additive contg. aminoacid as assimilable source of nitrogen - Google Patents

Abstract

A biodegradation additive contains (a) an assimilable source of N comprising (substd.) aminoacid, and (b) a source of P, with a ratio of N/P of 2-100. The additive has been treated to render it oleophilic.The N source forms at least 5 wt.% of the additive and comprises lysine, methionine, cystine, threonine, tryptophan, hydroxylysine and/or hydroxyproline. It may be in proteins forming at least 50 wt.% of the additive.

Description

 The present invention relates to a new biodegradation additive and its application for the treatment of environments contaminated by hydrocarbons, by acceleration of natural biodegradation.

 There are numerous methods using cultures of microorganisms, which are developed under controlled conditions (in reactors for example) and then applied to the medium to be treated. But these methods are not effective when it comes to working in an open environment.

There are indeed problems related to the dilution of microorganisms in the natural environment, and competition problems with native microorganisms much better suited to the environment considered. This is why we turned to the solution in which we stimulate the native micro-organisms by providing them with nutrients necessary for their development and which are limiting in the natural environment.

 Also, we have proposed nutritional additives which are added to the medium to be treated. These additives can be fertilizers of the type used in agriculture, or synthetic protein products, or even bacterial lyophilisates with the nutrient. These products meet the carbon, nitrogen and phosphorus needs of bacteria. The specific requirements of microorganisms for nitrogen and phosphorus correspond to a variable N / P molar ratio to a large extent, without appreciable alteration in efficiency. In addition to nitrogen and phosphorus, these additives contain assimilable carbon. This assimilable carbon is contained in hydrocarbon molecules whose chain is similar to an aliphatic chain which one meets in hydrocarbons. Moreover, these products serve as "starter", that is to say that they favor the reaction in the first moments.

 The availability of nutrients is also an important problem, since this conditions the kinetics of degradation of hydrocarbons. In order to accelerate this kinetics, various solutions have been proposed, which consist in mixing the nutrients with various additives and in forming suspensions, and especially emulsions. FR-A-2 490 672 describes microemulsions in which the nutrients are in an aqueous solution which is microemulsified in a lipomiscible liquid. However, this technique involves the step of forming the microemulsion and requires the presence of additives such as surfactants and the like, which are expensive. FR-A-2,512,057 describes an improvement of the solution proposed in the patent previously cited which consists in providing the nitrogen source in the form of a dual system comprising two chemically different kinds of nitrogen compounds. A preferred system is a system consisting of urea and amino acids. Furthermore, this patent teaches that amino acids alone are not as effective as the dual system. This dual system is also a microemulsion, it suffers from the same drawbacks as any (micro) emulsion.

 However, these synthetic additives present toxicity problems, due to the presence of derivatives, such as, for example, butoxyethanol, and other similar products.

 So we are looking for natural products as additives; but the problem is that these products do not contain carbon in a form close to an aliphatic group of the type of those present in hydrocarbons and, therefore, are not liable to lead to a choke effect.

Furthermore, additives are sought which can be used without requiring a (micro) emulsion or the use of expensive additives.

 We also know the use of animal meal as a nutrient for microorganisms, these being in aqueous solution or suspension.

Biosis Previews Databank. Philadelphia, Biosis Number: 83042449, GA Kochkina et al .: "Development of a Nutrient
Medium for Cultivating Ntomophthora-Thaxteriana "& Biotekhnologiya, vol. 4, 1986, pages 46-51, describes the use of fishmeal as a nutrient for the cultivation of a microorganism, for example Entomophthora-Thaxteriana fungi.

 Biosis Previews Databank, Philadelphia, Biosis Number: 70050436, M. Rusan et al .: "Influence of Animal Proteins on the Fermentation of Antibiotics" & Bol Soc Broteriana 52, vol. 0, 1978, (Recd. 1979), pages 29-36, describes the use of meat and blood meal as a source of nitrogen for the production of antibiotics from fungi-type microorganisms.

Biosis Previews Databank, Philadelphia, Biosis Number: 70043472, O. Yagi et al .: "Degradation of Poly Chlorinated Bi
Phenyls by Microorganisms "& J. Water Pollut Control Fed 52, vol. 5, 1980, pages 1035-1043, describes the use of microorganisms for pollution control, with the meat extract being added to the culture medium.

 However, there is no indication that these same flours can also be used in a medium contaminated by a hydrocarbon, that is to say as different as a simple aqueous medium.

 Surprisingly, the applicant has found that the additive according to the present invention meets all the requirements set out above.

 Thus, the subject of the present invention is a biodegradation additive characterized in that it consists of at least one animal flour, said flour comprising at least one assimilable nitrogen source consisting of at least one amino acid.

 By biodegradation is meant degradation by a microorganism, present in situ or reported. This application can therefore be carried out in an open environment in the presence of an indigenous bacterial flora, or on soils in the presence of an added specific bacterial flora, if that in presence is deemed insufficient.

 The microorganism used can be a yeast, a fungus, a bacterium; in fact any microorganism capable of degrading a hydrocarbon is appropriate. By way of non-limiting example, mention may be made of Pseudomonas, Acitenobacter, Flavobacterium, Artrobacter, Corynebacterium.

 By assimilable nitrogen is meant the nitrogen effectively metabolized by the micro-organism during degradation.

 The amino acids which can be used within the framework of the present invention can be any amino acid, natural or close synthetic acids such as ornithine, and the like. These amino acids can be unsubstituted or substituted. When substituted, the substituent is an alkyl, lower alkoxy, hydroxy and the like.

Preferably, the amino acid is chosen from the group consisting of: lysine, methionine, cystine, threonine, tryptophan, hydroxylysine, hydroxyproline, and mixtures thereof.

 Preferably, the assimilable nitrogen source represents at least 5% by weight of the total weight of said biodegradation additive.

 According to an embodiment of the present invention, the source of assimilable nitrogen is found in proteins representing at least 50% by weight of the total weight of said additive.

Alternatively, the flour is fish meal.

According to another variant, the flour is a meat meal.

 Fish or meat meal is obtained by any conventional manufacturing process. By way of example, mention may be made of the following process for meat meal: butchering of corpses, grinding thereof, sizing, preheating, draining, drying, press and final grinding. By way of example for fishmeal, the following process may be mentioned: shredding, cooking, press, mixing with a press juice concentrate, drying, sieving, and final grinding.

 The composition of these flours can vary to a large extent; as a representative but non-limiting example, compositions for various flours are given below.

Fish meal: protein: 60 to 85%
of which main amino acids: lysine, methionine, threonine.

fat: 3 to 25%.

mineral matter (phosphorus, calcium, chlorides): 5 to 24%,
Meat meal: protein: 60 to 85%
of which main amino acids: lysine, threonine, hydroxyproline.

fat: 2 to 7.

mineral matter (phosphorus, calcium, chlorides): 7 to 28%,
The use of additives according to the present invention, such as meat or fish meal, is therefore useful for the biodegradation of hydrocarbons on soils, sediments and on the surface of water. Sediments contaminated by hydrocarbons can come from accidental oil spills or not, such as cleaning of basins, pavement, soil, ...

This use is just as suitable and profitable, in the case of treatment in closed environments, such as reactors, quagmire, storage of hydrocarbons and others.

 The proportion of additive relative to the hydrocarbon is variable. The radditive] / hydrocarbons] mass ratio is generally between 3 and 30. Preferably, the mass ratio is equal to approximately 10.

 The present invention also relates to the use of the present additives for the biodegradation of hydrocarbons.

 The following examples illustrate the invention in more detail, without however limiting it.

EXAMPLES
COMPOSITION OF ADDITIVES
The composition of the biodegradation additives is given in the table below.

Products% Nitrogen% Phosphorus N / P% Carbon
(N) (P) (C). fish meal ... 12.1 0.6 20.2 50.1
(Solatlante G). meat meal 60% ... 9.8 3.95 2.5 35.4
(from Viandor extraction). meat meal 70% ... 12.0 3.1 3.9 40.8
(Creton from Viandor extraction). meat meal 80% ... 12.9 1.4 9.2 40.5
(Viandor extraction creton)
These flours have the following average analytical characteristics.

FISH MEAT60% MEAT70; MEAT 80%
PROTEINS 70-73 60 70 80
Lysine 5.20 3.25 4.20 4.80
Methionine 2.05 o, 85 1.00 1.20
Cystine 0.55 0.85 0.42 0.48
Threonine 2.65 2.00 2.40 2.75
Tryptophan 0.70 0.45 0.70 1.10
Hydroxylysine - 0.45 0.42 0.48
Hydroxyproline - 3.42 3.85 4.40
MOISTURE 3-5 4-9 3-7 3-7
FAT MATERIALS 20-23 2-6 2-4 2-4
MINERAL MATERIALS 5-7 25-28 17-20 7-10
Phosphorus 0.40-0.80 3.5-4.2 2.9-3.3 1.2-1.6
Calcium 0.15-0.50 7-9 5.5-7.0 2.4-3.7
Chlorides (NaCl) 2-3 1.4-1.6 1.1-1.5 0.8-1.0
EXAMPLE 1
Acceleration of biodegradation of hydrocarbons in a controlled laboratory environment
The bacterial cultures are carried out in closed reactors, which are 250 ml Erlenmeyer flasks, continuously agitated using magnetic stirrers (200 rpm).

These Erlenmeyer flasks, previously sterilized, were closed with suitable caps. The bacterial flora used is a complex marine flora (1.4.104 bacteria / ml) taken from Biarritz. The different media used are as follows:
reactor no. 1. control without biodegradation additive
reactor no 2 .. fish meal
reactor no 3 .. meat meal with 70% protein
reactor no 4 .. meat meal with 60% protein
reactor no 5 .. meat meal with 80% protein
. reactor no 6 .. sterile control (100 mg / l HgCl2)
All the nutrient media, apart from the controls, are composed of:
. 0.5 g / l crude *
. 0.05 g / l of biodegradation additives (i.e. 10% / gross)
. 160 ml seawater ** (dilution water and inoculum)
* Gross: Arabian Light topped at 1500C: BAL 150
** Sea water: 2.2 mg N-NO3 / l, pH 7.8.

 The sterile control makes it possible to quantify the elimination due to evaporation. The control without biodegradation additive makes it possible to estimate the potential of complex marine flora to naturally degrade hydrocarbons.

 Biodegradation monitoring is obtained by analyzing the marine bacterial flora and the disappearance of aliphatic hydrocarbons.

The enumeration of the total heterotrophic bacteria is carried out on agar (MARINE AGAR DIFCO) after incubation for 7 days at 27 "C.

Monitoring of changes in the composition of hydrocarbons is obtained by analyzing the aliphatic fraction of the hydrocarbons; biodegradation is followed by the estimation of the C17 / pristane and C18 / phytane ratios. Since pristane and phytane are more difficult to biodegrade than C17 and C18 alkanes, changes in ratios can quantify the biodegradation of the aliphatic fraction of hydrocarbons.

(The residual crude oil is estimated by:
- extraction of the whole sample with chloroform
(100 ml), evaporation of chloroform on an evaporator
rotary, ie rotavapor,
- analysis of the hydrocarbons taken up in hexane in
gas chromatography. This last step
allows estimation of biodegradation by calculation
C17 / pristane and C18 / phytane ratios.)
The various media, placed in the reactors, are stirred for 18 days, at 20 ° C., in the dark. After 18 days, a bacterial count is carried out before stopping the reaction by adding chloroform for analysis of the residual crude.

 Bacterial development is shown in Table I. The reactors to which a biodegradation additive has been added have a bacterial development greater than the control after 18 days of incubation.

TABLE I
Value of bacterial counts performed
after 18 days of culture
Additives Total bacteria / ml
Day O Day 18
. witness ... 1.4.104 3.5.104
. fish meal ... 1.4.104 5.3.105
. meat meal 60% ... 1.4.104 1.105
. meat meal 70% .. 1.4.10 4.7.105
. meat meal 80% .. 1.4.10 4.6.105
sterile witness ... O 0
The amount of crude removed by biodegradation is shown in Table II.

TABLE II
Elimination of the aliphatic fraction
hydrocarbons by biodegradation
Additives Elimination by biodegradation
. witness ... 0%
. fish meal ... 38.7%
. meat meal 60% .. 49.8%
. meat meal 70% .. 44.7%
. meat meal 80% ... 46.2%
. sterile witness ... 0%
Both the bacterial development and the biodegradation of BAL 150 are accelerated.

EXAMPLES 2 to 3
Accelerating the biodegradation of hydrocarbons in polluted soil by adding biodegradation additives (animal meal), with or without aeration
In order to test the use of animal meal under in situ treatment conditions, two series of tests are carried out in the treatment of soil polluted by hydrocarbons: the first is the treatment of a soil by adding biodegradation additives, the second is the same treatment to which a mechanical ventilation treatment has been added.

First test
The treatment of soil contaminated with hydrocarbons consists of adding animal meal to the polluted soil.

This animal meal allows the development of bacteria already present on the site and facilitates the biodegradation of hydrocarbons.

 In this test, a meat meal (60% protein: see composition example 1) is tested as a biodegradation additive for the treatment of plots of soil (1 m2) soiled with hydrocarbons. A treated plot and a control plot (without adding flour) are monitored for 2 months.

The soil moisture level is controlled and maintained at 15-20 * for the two months.

 The spreading of crude oil (Arabian Light topped at 150 C: BAL 150) is carried out, at a rate of 3 kg / m2, two months before application of the treatment. The quantity of flour applied is 225 g / m2 (7.5% compared to hydrocarbons). The samples were taken by coring over 20 cm, at 0, 7, 15, 30 and 60 days.

 Biodegradation monitoring is carried out by analyzing the bacterial flora of the soil and the disappearance of hydrocarbons by biodegradation.

 The count of aerobic heterotrophic total bacteria is carried out on agar (glucose medium) after incubation for 7 days at 27 "C. The count of the specific hydrocarbon flora is carried out on liquid medium by the technique of the most probable number (incubation at 27" C for 21 days).

 The estimation of the quantity of residual crude oil is carried out as described in Example 1, namely by the analysis of the C17 / pristane and C18 / phytane ratios.

 The results obtained for the analysis of the bacterial flora are presented in Table III.

TABLE III
Temporal evolution of total bacterial flora
aerobic and specific hydrocarbons
Control days Plot treated
with flour
Total heterotrophic aerobic bacteria / g dry sediment
0 6.4.104 5.6.104
7 1.1.105 6.9.105
15 4.2.104 2.2.105
30 8.5.105 5.3.106
60 1.6.105 2.6.105
Control days Plot treated
with flour
Specific hydrocarbon bacteria / g dry sediment
0 2.5.103 5.6.104
7 2.8.104 6.9.1or
15 1.104 2.2.105
30 2.3.105 5.3.106
60 4.8.104 2.6.105
The temporal evolution of bacteria, total and specific hydrocarbons, in the plot treated with animal meal shows a greater development than in the control plot. This shows that the bacterial activity in the treated plot is higher, it is stimulated by the presence of animal meal.

 The biodegradation results of aliphatic hydrocarbons are presented in Table IV.

TABLE IV
Biodegradation rate (t) of aliphatic hydrocarbons
Control days Plot treated
with flour biodegradation rate of aliphatic hydrocarbons g
OOO
7 7.5 0
15 17.0 29.0
30 21.0 53.0
60 28.0 53.0
The results indicate a clear difference between the biodegradation observed in the control plot and in the plot treated with flour. Meat meal therefore stimulates the biodegradation of hydrocarbons on polluted soil without the addition of bacteria.

Second test
It is a similar test with this time adding a mechanical ventilation treatment.

 During this test, we used an oily mud containing 23% of hydrocarbons. The flour is brought in at a rate of 5% by weight of hydrocarbons at the time of spreading and a new supply takes place six weeks later.

Three types of animal meal are tested; 70% and 80% meat and fish (composition: see example 1). The treated plots are compared to a control plot. The duration of the experiment is two months. For each plot, an average sample is made from six samples to a depth of 10 cm. The samples are taken every week for 8 weeks then weeks 10, 14, 18 from the start of the experiment. Aeration is practiced the fourth week.

 The evaluation of the total and specific aerobic bacterial population is made by counting colonies isolated on a Petri dish (glucose medium and medium 90% petrol; 10% gas oil). The volatile fraction of the medium is evaluated by distillation of the samples taken from the sludge alone plot. The determination of residual hydrocarbons is carried out by gravimetry on the dry extract of each sample. The rate of biodegradation of the hydrocarbons is determined after passage through gas chromatography of the alkane fraction.

 The results of the evolution of the total heterotrophic aerobic and specific hydrocarbon bacterial flora are reported in Table V.

TABLE V
Evolution of the total aerobic and specific hydrocarbon bacterial flora on soil contaminated with hydrocarbons after treatment with different animal meal or untreated, and with mechanical aeration of the soil
A second addition of flour is made in week 6.

<tb> Week <SEP> Control <SEP> Meat <SEP> 70% <SEP> Meat <SEP> 80% <SEP> Fish
<tb>

Total aerobic bacteria / g of dry sediment 0 2.00E + 05 1.50E + 06 2.80E + 05 3.30E + 05 1 8.60E + 04 1.50E + 07 1.10E + 08 4.80E + 07 2 2.40E + 05 1.40E + 08 2.10E + 08 7.20E + 07 3 7.30E + 05 1.70E + 08 1.80E + 08 1.80E + 08 4 5.20E + 05 6, 40E + 07 8.30E + 07 3.70E + 07 5 3.10E + 05 5.60E + 07 4.20E + 07 5.30E + 07
6 1.40E + 05 1.50E + 07 9.90E + 07 3.20E + 07 7 4.40E + 05 2.60E + 08 2.40E + 08 5.30E + 08 8 7.00E + 05 3, 50E + 08 5.30E + 08 4.80E + 08 10 6.40E + 04 9.20E + 07 1.20E + 08 1.80E + 08 14 4.90E + 04 7.90E + 07 1.30E + 08 1.30E + 08 18 1.20E + 05 8.30E + 07 1.40E + 08 4.90E + 07
Specific bacteria / g of dry sediment 0 2.40E + 03 5.10E + 04 9.90E + 03 9.40E + 03 1 9.60E + 03 4.00E + 06 2.40E + 07 6.00E + 06 2 3.40E + 03 7.20E + 07 7.20E + 07 2.90E + 07 3 9.30E + 03 1.60E + Ç) 7 4.80E + 07 3.60E + 07 4 5.00E + 03 6 , 20E + 06 1.30E + 07 7.80E + 06 5 4.60E + 03 3.80E + 06 6.70E + 06 1.90E + 07
6 1.40E + 03 3.10E + 06 1.10E + 07 1.20E + 07 7 2.90E + 03 6.50E + 07 1.90E + 08 3.70E + 08 8 5.10E + 04 1, 00E + 08 1.10E + 08 1.10E + 08 10 1.40E + 03 1.40E + 07 6.50E + 06 3.30E + 07 14 4.10E + 03 1.70E + 07 8.40E + 06 2.90E + 07 18 6.20E + 03 6.20E + 06 5.80E + 06 6.90E + 07
We observe a rapid growth the first two weeks, then the flora decreases. Aeration and the second addition of flour cause new growth.

 The bacterial developments observed in the presence of animal meal are much higher than those observed in the control plot (sludge). This indicates that animal meal stimulates bacterial flora.

 The results obtained for the evaluation of the biodegradation are presented in Table VI.

TABLE VI
Biodegradation rate (%) of aliphatic hydrocarbons
in soil polluted by hydrocarbons, treated and untreated
Witness Week Meat 70% Meat 80% Fish
Biodegradation rate of aliphatic hydrocarbons%
O 0 OOO
1 - - 12.6 30
2 6.1 17.1
3 - 25.9 - 42.2
4 12.4 - 43.7
5 - - 52.4 60.3
6 35.2 80.3 70.9
7 - - 78.6 84.4
8 65.2 93.3 77.7 84.8
The study of the chromatograms makes it possible to observe the disappearance of the aliphatic hydrocarbons indicative of the biodegradation. The evolution of the biodegradation rate over time shows that meat and fish meal have an accelerating effect on biodegradation.

 Indeed, after four weeks of spreading, the biodegradation rate for meat and fish meal is around 40% while it is only 10% for the sludge control alone.

 The application of animal meal on soil contaminated by hydrocarbons therefore accelerates the biodegradation of hydrocarbons.

EXAMPLE 4
Acceleration of the biodegradation of hydrocarbons from contaminated sandy sediments in the supratidal zone
The study of the biodegradation of hydrocarbons on sand was carried out in an open environment in basins of 600 liters in which a beach of sand was reconstituted. The basins are supplied with daily water renewal from a nearby pumping station.

 After allowing the system to stabilize for 1 month, the sand is soiled with 2 liters of BAL 150 hydrocarbon (Arabian Light etêtée at 1500C) per m2 of sand and left to stand for one week before treatment. One basin was treated with animal flour (10% by weight of hydrocarbon; meat flour at 60% protein, see composition example 1), another served as a control.

 Bacteriological monitoring (total aerobic heterotrophic bacteria and specific hydrocarbon bacteria) is carried out on the sediment. The number of bacteria is reduced to the weight of dry sediment. Aerobic heterotrophic marine bacteria are counted on Zobell-2216E agar medium. The specific hydrocarbon marine bacteria are counted in liquid medium by the technique of the most probable number and consultation of the tables of MC CRADY.

 The moisture content of the sand is 17.0% + 0.5%. Hydrocarbons are also monitored in the sediment. The estimation of the quantity of residual crude oil is carried out as described in Example 1, namely by the analysis of the C17 / pristane and C18 / phytane ratios.

 The sediments are sampled at times: 0, 7, 15, 30, 45 and 60 days. The average outside temperature can reach 30 "C during the day. The average water temperature is 25" C, its salinity 40/00 and its pH 8.

The experiment is conducted without a tide, which means that biodegradation is studied in a supratidal area.

Different core samples allow us to observe that the crude enters the first 5 centimeters of sediment. I1 therefore remains in the supratidal zone where the oxygenation conditions are correct.

 The results obtained for the analysis of the bacterial flora are presented in Table VII.

TABLE VII
Temporal evolution of total bacterial flora
aerobic and specific hydrocarbons in the area
supratidal of a sandy sediment
Control days Plot treated
with flour
Total aerobic bacteria / g wet sediment
0 2.0.107 2.3.107
7 2.4.107 7.6.107 9
15 1.9.107 2.8.109
30 5.7.107 4.8.108
45 1.0.108 8.9.108
60 4.7.107 6.8.108
Specific hydrocarbon bacteria / g of wet sediment
0 1.1.104 1.1.104
7 4.6.105 1.1.106
15 4.6.105 1.1.106
30 2.1.105 3.9.106
45 1.5.106 8.9.106 60 2.1.106
The temporal evolution of bacteria, total and specific hydrocarbons, in the plot treated with animal meal shows a more significant development than in the control plot. This shows that in the supratidal zone of a sandy sediment, the bacterial activity in the treated plot is greater than that untreated. It is stimulated by the presence of animal meal.

 The biodegradation results of aliphatic hydrocarbons are presented in Table VIII.

TABLE VIII
Biodegradation rate (e) of aliphatic hydrocarbons
in the supratidal zone of a sandy sediment,
at 60 days
Control days Plot treated
with flour biodegradation rate of aliphatic hydrocarbons%
OOO
60 25.0 38.0
EXAMPLE 5
Acceleration of the biodegradation of oil in contaminated sandy sediments in the intertidal zone
The experiment was carried out under the same operating conditions as in Example 4, apart from the climatic conditions which are different (winter conditions) and the time of the experiment which lasted longer.

 Sediments are sampled at times: 0, 7, 15, 30, 45, 60, 75 and 90 days. The average outside temperature can reach 30 "C during the day. The average water temperature being 20" C, its salinity of 400 / oc and its pH of 8. The experiment was carried out with a tidal simulation, which means that biodegradation is studied in a supratidal and intertidal zone.

 The results obtained for the analysis of the bacterial flora are presented in Table IX.

TABLE IX
Temporal evolution of aerobic heterotrophic and specific hydrocarbon bacterial flora in the supratidal, intertidal and submerged areas of a sandy sediment.

Sample days Plot treated with flour
Zone Zone
supra- inter- immer- supra- inter- immer
tidale tidale gée tidale tidale gée
Total aerobic bacteria / g wet sediment
0 1.6.107 1.3.107 2.2.106 1.6.107 1.3.107 2.2.106
7 3.2.107 1.0.107 2.7.106 7.4.107 4.9.107 4.6.106
15 4.1.107 1.9.10.6 1.3.108 4.4.107 4.8.106
30 2.1.107 2.0.107 1.0.106 2.3.108 2.3.107 7.2.105
37 1.5.107 3.0.106 5.1.105 2.4.108 6.0.107 1.0.106 45 2.7.107 1.9.107 3.4.106 7.8.108 6.2.107 5.9.106
7 7 6 8 8 6
58 5.4.107 3.2.10 '2.2.106 3.3.108 2.1.108 9.3.106
89 4.1.107 4.8.1o6 1.6.106 8.9.107 1.1.108 9.5.106
Specific hydrocarbon bacteria / g wet sediment
0 - - 4.6.104 - - 4.6.104
7 1.1.107 4.6.106 2.4.104 4.6.107 1.1.108
15 2.4.107 4.6.107 4.6.104 9.3.107 2.3.107 1.1.105 30 9.3.106 - 4.3.104 2.4.108 2.4.107 9.3.104
6 6 3 8 7 5
37 4.3.106 2.3.106 9.3.103 2.4.108 2.4.107 2.4.105 45 9.3.106 - 4.3.104 1.1.108 3.0.107 2.4.105 58 4.3.106 4.5.105 1.5.103 2.0.107 4 , 6.107 1.106
6 5 6 7 7 6
89 9.3.106 2.3.105 4.6.104 4.3.107 4.6.1o7 2.4.106
The temporal evolution of heterotrophic aerobic bacteria and specific hydrocarbons, in the plot treated with animal meal shows a more significant development than in the control plot, this for all the zones: supratidal, intertidal and submerged, while the submerged zone does not has not been polluted by oil and has not been treated.

 The biodegradation results of aliphatic hydrocarbons are presented in Table X.

PAINTINGS
Biodegradation rate () of aliphatic hydrocarbons
in the supratidal and intertidal zones of a
treated and untreated sandy sediment
Sample days Plot treated with flour
Zone Zone
intertidal supratidal intertidal supratidal
OOOOO
7 7 - 17 1 15 0 19 25 24 30 13 18 - 22 45 18 - 32 60 23 27 27 60 75 36 22 40 50
The results obtained show that animal meal stimulates the biodegradation of hydrocarbons in the supratidal and intertidal zone of a sandy sediment contaminated by hydrocarbons.

Claims (7)

 1.- Biodegradation additive characterized in that it consists of at least one animal flour, said flour comprising at least one source of assimilable nitrogen consisting of at least one amino acid.  2.- Additive according to claim 1, characterized in that it consists of a fish meal.  3.- Additive according to claim 1, characterized in that it consists of a meat meal.  4.- Additive according to any one of claims 1 to 3, characterized in that the amino acid is chosen from the group consisting of: lysine, methionine, cystine, threonine, tryptophan, hydroxylysine, hydroxyproline, and mixtures thereof.  5.- Additive according to any one of claims 1 to 4, characterized in that the assimilable nitrogen source is in proteins representing at least 50% by weight of the total weight of said additive.  6.- Additive according to any one of claims 1 to 5, characterized in that said additive is present in a mass ratio [additive] / [hydrocarbons] between 3 and 30, and preferably equal to about 10.  7.- Use of an additive according to any one of claims 1 to 6, for the biodegradation of hydrocarbons.