MXPA99008133A - Biodegradation of oil sludge - Google Patents

Abstract

A method for the biodegradation of an oil-based sludge comprising a mixture of petroleum hydrocarbons is disclosed. The method comprises forming an aqueous solution in a reactor of an oil-in-water emulsion of the oil-based sludge, bacterial culture and nutrients for the bacterial culture, the bacterial culture having the ability to grow on petroleum hydrocarbons as sole carbon source and having been isolated from a hydrocarbon contaminated soil or hydrocarbon-containing sludge or other environments rich in hydrocarbon degrading bacteria, maintaining the aqueous solution under aerobic conditions in the reactor at a temperature of at least 10°C for a period of time sufficient to reduce the amount of hydrocarbon by at least 25%, and discharging aqueous solution having a reduced amount of hydrocarbons from the reactor. The method may be used on sludge containing aromatics, resins and asphaltenes.

Description

BLODEGRADA lON "DE LODOS OLEOSOS The present invention is directed to the treatment of oily sludges, and in particular to the biodegradation of oily sludge to environmentally acceptable products. As such, the present invention is directed to the treatment of compositions with high concentrations of total oil / sludge hydrocarbons, examples of which are oil sludge refinery, tank bottom sludge and oil storage tank vessels, waste sludge oily wells, the so-called treatment emulsions or sludge oil, oily sludge from solid processing containing oily waste including centrifuged sludge, clay fines, and drilling sludge. In contrast to waste water treatment processes using low concentrations of total petroleum hydrocarbons or processes for the production of simple cell protein, biomass or bacterial cells - The b.odecjradación of oily raw materials has been directed mainly to cleaning , that is, bio-remediation, of soils and edges I of beaches contaminated with oil, as a result of oil spills in the land of, for example, underground storage tanks, or oil tankers at sea. Such bioremediation of hydrocarbons generally involves the creation of conditions in the soil or on the edge of the beach that promote the growth of This is based on the use of petroleum hydrocarbons, facilitating the conversion of hydrocarbons to biomass and / or their degradation, ultimately to carbon dioxide and water. Hydrocarbons are the carbon source for microbial growth, although it may be necessary to add other ingredients, especially nitrogen and phosphorus, as fertilizers. Microorganisms also require a range of inorganic ions for growth, but such ions are usually present in adequate amounts in the soil that is being treated. The bio-remediation processes generally use aerobic microorganisms that require aeration / oxygenation by maximizing the contact of the contaminated material with atmospheric oxygen through soil cultivation or aeration procedures using positive and negative pressure air pumping systems. It is understood that the general hierarchy of microbial activity in crude oil is aIiphatics > aromatics > res > so, altenos Thus, high molecular weight and aromatic hydrocarbons are more difficult to degrade, compared to minor alkanes. Liquid-solid treatment systems have also been used to degrade petroleum hydrocarbons. However, long degradation treatment periods are found, for example, 50-100 days. The treatment of crude oil waste land and oil sludge refinery has been used for many years as a method of disposal of oil and mud. The rates of biodegradation and microbial growth tend to be suboptimal in soil culture processes and the process is not easily controlled. In addition, the process is influenced by the composition of the soil, climate and temperature, as well as the methods used for cultivation in the land cultivation processes. For large refineries, large areas of land have to be consigned for such a process, and even more, the first step in the process involves soil contamination with the oils to be degraded. U.S. Patent 3 899 376 discloses a single or multiple tank system that is directed primarily to wastewater treatment. The process uses a particular bacterial strain from a culture collection for the bio-remediation process. U.S. Patent 5,364,789 discloses a microbial cleaner comprising at least one microbial strain that ingests hydrocarbons and a biocatalyst that transforms the hydrocarbons into non-toxic substances. The biocatalyst includes a non-ionic surfactant, a salt that absorbs chlorine, at least one nutrient of microbes and water. It is stated that the cleaner can be used in virtually any situation that requires the removal of hydrocarbons, including cleaning contaminated soil and treating oil spills in soil and water. U.S. Patent 5 271 845 discloses a process for treating hydrocarbon oil that is correctable to attack by extracellular hydrolytic enzymes, a property not shared by major petroleum hydrocarbon components. US Pat. No. 5,364,789 describes a microbial cleaner, which, among other applications, can be used to remove oily sludge. This cleaner is disadvantageous since high proportions of cleaning treatment are used.

JP-A-09038630 describes a system for the treatment of an oil suspension using a surfactant and bacteria, in which high amounts of bacterial products and surfactants are added. A method for the biodegradation of a slurry fraction of petroleum hydrocarbons has now been found, using such a reactor method. Accordingly, an aspect of the present invention provides a method for the biodegradation of a petroleum-based sludge, said petroleum-based sludge comprising a mixture of petroleum hydrocarbons, said method comprising the steps of: (a) forming a solution water in an oil-in-water emulsion reactor, bacterial culture and nutrients for said bacterial culture, said oil-in-water emulsion being an emulsion of said oil-in-water-based sludge, said bacterial culture having the capacity to grow in petroleum hydrocarbons as a single source of carbon and having been isolated from a soil contaminated with hydrocarbon or hydrocarbon-containing sludge or other environments rich in hydrocarbon-degrading bacteria, using microbial enrichment techniques using hydrocarbons in the selection medium, said reactor containing 50% by volume of total petroleum hydrocarbons; (b) maintaining said aqueous solution under aerobic conditions in the reactor at a temperature of at least 1 ° C for a period sufficient to reduce the amount of hydrocarbons by at least 25%, and at a favorable pH for the promotion of bacterial growth and degradation of hydrocarbons; and (c) discharging the aqueous solution having a reduced amount of total petroleum hydrocarbons from said reactor. In a preferred embodiment of the present invention, the nutrients comprise bioavailable compounds of nitrogen, phosphorus and potassium, especially in which the nitrogen compound is an organic ammonium, nitrate or nitrogen ion, and phosphorus is phosphate. In another embodiment, the reactor contains about 5-50% by volume of total petroleum hydrocarbons, especially about 10-30% by volume of total petroleum hydrocarbons. The oil-based sludge preferably contains extractable hydrocarbons with hexane in an amount in the range of up to 500,000 ppm, especially in the range of 65,000-250,000 ppm. For clarification, the term total petroleum hydrocarbons (or TPHs) as used herein, is defined as extractable hydrocarbons with hexane and hydrocarbons soluble in hexane. In yet another embodiment, the nutrients are in proportions corresponding to relative proportions in bacterial cells, and are supplied at concentrations which promote high levels of bacterial growth and high rates of hydrocarbon degradation. In additional modalities, petroleum hydrocarbons consist of mixtures of saturated hydrocarbons, aromatic hydrocarbons, hydrocarbon resins and asphaltenes, especially petroleum hydrocarbons obtained from oil refinery sludges, from the bottom of an oil storage tank, from a petroleum well on land or the washing of a hold on a tanker. In other embodiments, the amount of nitrogen required to support the process is in the range of 50-1000 ppm, and preferably in the range of 300-700 ppm, and the minimum amount of phosphate is in the range of 10-200 ppm. and preferably 50-1 50 ppm. In additional embodiments, the aqueous solution contains a surfactant, more especially a non-ionic or an anionic surfactant. The surfactant is in an amount sufficient to form said oil-in-water emulsion, especially in which the amount of surfactant is less than 2500 ppm and preferably less than 1500 ppm. It is preferred that the ratio of the amount of petroleum hydrocarbons to surfactant is at least 40: 1. In additional embodiments, the aqueous solution is treated in the reactor for a holding time of at least 7 days. In other modalities, the sludge is chemically or physically pretreated to improve biodegradability before, or during, biodegradation. A further aspect of the invention provides a method for the biodegradation of a petroleum-based mud, said oil-based mud comprises a mixture of petroleum hydrocarbons, said method comprising the steps of: (a) forming an aqueous solution in a reactor of an oil-in-water emulsion, bacterial culture and nutrients for said bacterial culture, said oil-in-water emulsion being an emulsion of said oil-in-water-based emulsion, said bacterial culture having the capacity to grow in petroleum hydrocarbons as a source unique carbon and being native bacteria in the oil-based mud, multiplying such native bacteria and degrading the mud, said reactor containing up to 50% by volume of total petroleum hydrocarbons; (b) maintaining said aqueous solution under aerobic conditions in the reactor at a temperature of at least 10 ° C for a period sufficient to reduce the amount of hydrocarbons by at least 25%, and at a favorable pH for the promotion of bacterial growth and degradation of hydrocarbons; and (c) discharging the aqueous solution having a reduced amount of said hydrocarbons from said reactor. The method of the present invention relates to the biodegradation of a petroleum-based mud. Oil-based sludge comprises a mixture of petroleum hydrocarbons and may include liquid or solid contaminants other than oil and water. The mixture of petroleum hydrocarbons would normally comprise a mixture of aliphatic hydrocarbons, aromatic hydrocarbons, hydrocarbon resins and asphaltenes.

The present invention is particularly directed to the biodegradation of a mixture of petroleum hydrocarbons from aliphatics, aromatics, resins and asphaltenes. Such mixtures of petroleum hydrocarbons can be obtained from a variety of sources. For example, the mixture may be in the form of a sludge obtained from an oil refinery. The mud can also be obtained from the bottom of a storage tank that has been used for the storage of petroleum oil, the mud being obtained particularly when the storage tank is cleaned or drained. Alternatively, the hydrocarbon mixture could be an oil residue obtained from around a wellhead on the ground, be a clay fines material containing oil, or be the cleanup of a tanker's hold used for transportation. of petroleum products. The petroleum hydrocarbon mixture, which is referred to herein as a slurry, can also be obtained from a variety of other sources. In each case, the sludge is characterized as having a substantial proportion of heavy final petroleum hydrocarbons, which may require the use of a solubilizing agent or surfactant to facilitate mixing and dispersion in water, such as an oil-in-water emulsion. The method of the present invention is carried out in a reactor. It is preferred that the reactor be a simple stage reactor that is charged with the solution described herein, which is allowed to incubate for a period to reduce the amount of hydrocarbons within the aqueous solution, and then is subsequently discharged from the reactor. However, it will be understood that the reactor would be in the form of a series of reactors in which the aqueous solution is passed from reactor to reactor before being finally discharged from the process for the biodegradation of the sludge. In the method, an aqueous solution is fed to the reactor. The aqueous solution is comprised of an oil-in-water emulsion, bacterial culture and nutrients for the bacterial culture. The mud is in the form of the oil-in-water emulsion. The amount of petroleum hydrocarbons fed to the reactor is governed primarily by the formation of the oil-in-water emulsion. In particular, the aqueous solution may contain up to 50% by volume of total petroleum hydrocarbons, depending on the particular hydrocarbons. In preferred embodiments, the reactor contains 5-50% by volume of total petroleum hydrocarbons, especially 10-30% by volume. Oil-based mud contains extractable hydrocarbons with hexane. In preferred embodiments, the amount of extractable hydrocarbons with hexane is up to 500,000 ppm, especially in the range of 65,000-250,000 ppm. It would normally be necessary to incorporate a surfactant into the aqueous solution and to subject the aqueous solution to agitation, in order to form the oil-in-water emulsion. The surfactant is preferably a nonionic or anionic surfactant and is used in an amount sufficient to form the emulsion. However, the amount of the surfactant is preferably less than 2500 and particularly less than 1500 ppm.

In addition, the amount of surfactant, if added, is maintained at a level as low as is consistent with obtaining the oil-pet emulsion. In particular, it is preferred that the ratio of petroleum hydrocarbon to surfactant be at least 40: 1, and especially at least 60: 1. The aqueous solution also contains a bacterial culture. The bacterial culture used in the method of the present invention is a bacterial culture that occurs naturally. Such a culture can be isolated from a soil contaminated with hydrocarbons or mud containing hydrocarbons or from other environments, including soil or activated iodine, which can be rich in hydrocarbon degrading bacteria, and inoculated in a basal medium, as described in the present. The bacterial culture is selected for its ability to grow in petroleum hydrocarbons as the predominant source of carbon in the basal medium. Bacterial enrichment techniques for isolation of a bacterial culture capable of growing in hydrocarbons is well understood in the art. Typical techniques include adding a soil sample, mud or other material containing a large population of bacteria to an aqueous medium containing hydrocarbons as the sole or predominant source of carbon. Other chemical components are also added, including a source of inorganic nitrogen, phosphorus and salts needed to support bacterial growth. Such a medium can be used to preferentially promote the multiplication of hydrocarbon degrading bacteria using standard aerobic microbial cultivation methods, including incubation in aerated microbial culture vessels. An efficient hydrocarbon degrading culture is selected by transferring a small amount of the resulting growth culture to additional samples of the same medium and repeating the process one or more times. The culture can be maintained or stored using methods well known in the art. In order to prepare a high density culture for use as an inoculum for sludge degradation, the maintained culture can be inoculated into an aqueous medium consisting of the nutrients described herein, supplemented with petroleum hydrocarbons and incubated in a aerated reactor or fermentor or other culture vessel. The preferred inoculum volume is 0.1-20% by volume of total culture volume, preferably 1-5% by volume. The preferred concentration of petroleum hydrocarbons used in this inoculum development medium is 0.5-5%, and can be obtained from various sources including oil sludge, crude oils or refined oils, such as diesel. A typical aeration rate of the inoculum reactor is 0.1 -1.0 volumes of air per volume of medium per minute, with the culture incubated in the temperature range 20-37 ° C for 1 -7 days, preferably at 27-7 days. 33 ° C, at a pH generally maintained in the range 6.5-8.0, preferably in the range 7-7.5. The resulting bacterial culture can be used to inoculate the reactor containing the sludge to be degraded, at a rate of 0.1-20% total sludge volume, preferably 1-5%. Where a much larger volume of inoculum is required, the resulting inoculum can be transferred as an inoculum to a larger culture vessel and the culture development process is repeated on the larger scale.

The aqueous solution fed to the reactor also contains nutrients for the bacterial culture. A wide variety of nutrients can be used for bacterial culture, as will be understood by persons skilled in the art. Such nutrients will include nitrogen, phosphorus and potassium compounds, and would normally also include a variety of other ingredients. In particular, the nutrients comprise bioavailable compounds of nitrogen and phosphorus. In embodiments, the amount of nitrogen is in the range of 50-1000 ppm and preferably 400-700 ppm, and the amount of phosphate is in the range of 10-200 ppm, and preferably 50-150 ppm. In addition to the nitrogen and phosphorus compounds, the nutrient also contains optimized concentrations of compounds other than nitrogen, phosphorus, carbon, oxygen and sodium, required to support bacterial growth and therefore, it is usually necessary to add one or more of the magnesium, manganese, inorganic or organic sulfur, calcium, iron, copper, cobalt, zinc, boron and molybdenum. It will be appreciated that a guide for the selection of the relative amounts of nitrogen, phosphorus and other nutrients required relates the concentrations to the amounts of these components present in the bacterial cells. By providing an appropriate balance of nutrients and by adjusting nutrient concentration, it is possible to achieve high growth levels of hydrocarbon degrading bacteria and thus, accelerated rates of hydrocarbon degradation. For example, Greasham (1 993) "Biotechnology, a multivolume comprehensive treatise" (Biotechnology, an extensive multi-volume treatise "(Eds, Rehm, HJ, et al) Vol. 3, p. 31, VCH, Weinheim) has reported that the elemental non-carbon composition of major bacterial components is 12.5% nitrogen, phosphorus 2.5%, potassium 2.5%, sodium 0.8%, sulfur 0.6%, calcium 0.6%, magnesium 0.3%, copper , 0.02%, manganese, 0.01% and iron, 0.01%) .The use of appropriate concentrations and proportions of nutrients has to avoid a situation where growth is limited by suppression of an essential nutrient, while all other nutrients may be present in excess, the hydrocarbon provides the carbon source for growth, the oxygen is obtained by aeration of the crop, sodium is provided in the form of caustic soda, required to adjust the pH, it is also understood that in some cases, some of these nutri components can be present in sufficient quantities in some oil sludge or water added, so that the addition of selected nutrients may not be required in some cases. A disadvantage of depending on the nutrients present as contaminants in the mud or water is that their concentrations can be variable, thus introducing inconsistencies in the process. An example of a nutrient composition is as follows: N as NH, NO3, or organic N 500-700 ppm P as phosphate or related form 100-120 ppm K 50-90 ppm Mg 10 ppm Mn 1-4 ppm S as sulphate or organic sulfur 15 ppm Ca 8-1 2 ppm Ferric ion 1 ppm Copper 0.5 ppm Surfactant (nonionic or anionic) 1250 ppm Co, Zn, B, Mo 5-10 ppb each The relative proportions of these nutrients are similar to those proportions normally found in the compositions of bacterial cells. Other examples of nutrient compositions are given in the Examples hereinafter. The aqueous solution in the reactor is maintained at a temperature of at least 10 ° C. Preferred temperatures are 1 5-37 ° C, and especially 20-33 ° C. The aqueous solution is kept in the reactor for a period sufficient to reduce the amount of total petroleum hydrocarbon is 5-20 days, depending on the petroleum hydrocarbon being treated and the reactor conditions. Subsequent to the maintenance of the aqueous solution at the predetermined temperature for a period, the aqueous solution is discharged from the reactor. The aqueous solution has a reduced amount of hydrocarbons, including a reduced amount of the hydrocarbons of the group comprising the aromatics, resins and asphaltenes. The present invention can be used for sludge biodegradation, as described herein. In particular, it can be used for the biodegradation of a combination of hydrocarbon components from the fractions: saturated, aromatic, resins and asphaltenes.

It can also be used to preferentially degrade a proportion of the hydrocarbons, in a manner which causes the emulsion to break and facilitate the separation of a water phase and a residual oil phase. The oil phase can be reclaimed for reuse. Alternatively, the oil phase can be recycled to the next reactor cycle with only the aqueous phase of the reactor being discharged. The aqueous phase contains high concentrations of hydrocarbon degrading bacteria. In this way, the aqueous phase can be used for processes including soil bio-remediation processes, by direct atomization of the water in the contaminated soil. Alternatively, bacteria can be recovered from the aqueous phase by known methods (filtration or centrifugation) and subsequently, bacteria can be applied in these other processes. Where subsequent batches of sludge will be degraded in the reactor, a portion of the degraded sludge amounting to, for example, 1-20% of the reactor volume, can be retained in the reactor following discharge, as an inoculum source for the reactor. next batch of mud. In addition to the batch degradation process described above, it is envisaged that the invention will be extended to feed batch reactor processes, continuous or semi-continuous. In the batch feeding process, after the batch process has proceeded for some time, additional mud and / or nutrients / surfactants are added in one or more intervals and the process is allowed to continue. In continuous or semi-continuous processes, the degraded batch is removed from the reactor and replaced with undegraded sludge and nutrients / surfactants on a continuous basis or at intervals, respectively. The invention is illustrated by the following examples. Unless stated otherwise, all examples of the invention illustrated herein were conducted under non-sterile conditions. In addition, all of the biodegradation reactions exemplified herein, used oil-in-water emulsions.

EXAMPLE I The basal medium used in this example contained (by I): KH2PO4, 1.0 g; Na 2 H PO 4, 1.5 g; MgSO 4"7H 2 O, 0.2 g; Na2CO3, 0.1 g; CaCl2.2H2O, 0.05 g; FeSO4, 0.005 g; MnSO4, 0.02 g; and trace metal solution, 3 ml. The trace metal solution contained (by I): ZnCl2.4H2O, 0.0144 g; CoCl2, 0.012 g; Na2MoO4.2H2O, 0.012 g; CuSO4.5H2O, 1.9 g; H3BO4, 0.05 g; and HCl, 35 ml. The initial pH of the nutrient was adjusted to 7.2. A population of bacterial culture mixed in a cyclone fermentor with a work volume of one liter was maintained. Petroleum hydrocarbon degrading bacteria were selected for their ability to grow in petroleum hydrocarbons as the only carbon source in the basal medium described above. To initiate the selection of degrandante bacterial culture of petroleum hydrocarbons, a mixed population of bacteria, isolated from soil contaminated with hydrocarbons, was inoculated in basal medium supplemented with 2.0 g of NH4CI / I and 1.0 g of NaNO3 / l in the cyclone. Sludge A or B (60 g / l) was used as carbon source; the sludge is described later. It was found that the bacterial population reached 108 to 1010 CFU / ml in one week. Subsequently, the culture was maintained by removing 10% by volume from the reactor and replacing it with 10% by volume of fresh basal medium and sludge each day. Using this procedure, an actively growing crop was maintained. The sludge samples were collected from different ponds or lagoons located in different oil refineries. The content of total petroleum hydrocarbons (TPH) (extractables with hexane) was determined for each sludge. The composition of the different sludges is given in Table 1.

Table 1 Source of Soluble in Water Insoluble (%) Hexane (%) Hexane (%) Mud A 25 13 62 Mud B 13 3 84 Mud C 12 11 77 Mud D 65 15 20 Mud E 31 16 53 Mud F 22 6 72 Sludge G 89 11 0 EXAMPLE 11 The nutrient medium used for biodegradation in this example consisted of the basal medium supplemented with 2.0 g of urea / 1 and 1.0 g of yeast extract / l. The runs to determine the biodegradation of total petroleum hydrocarbons (TPH) with respect to the incubation time were carried out in 250 ml of Erlenmeyer flasks containing 10 ml of nutrient medium and 10 g of mud., giving a final sludge concentration of 50% in the total contents of the flask. The flasks were inoculated with 0.6 ml of mixed culture actively growing from the cyclone, maintained as described above, and incubated for 24 days at 25 ° C. The residual TPH content was determined as follows. At different time intervals, the contents of complete flasks were extracted with 40 ml of hexane and centrifuged at 10,000 rpm for 20 minutes. The hexane layer (top) was pipetted and transferred to a pre-weighed bottle. The hexane was allowed to evaporate in a vent hood and the residual oil was weighed to determine total petroleum hydrocarbons (TPH). The results are given in Table 2.

Table 2 Incubation time (days) Degradation of TPH (%) 6 32 14 37 18 47 24 48 It was found that over a period of 18 days, approximately 47% degradation of TPH occurred. No significant difference was obtained in degradation levels between 18 and 24 days.

EXAMPLE III In order to investigate the effect of surfactant on the biodegradation of TPH, 5 different surfactants were tested at 0.25% concentration. In each test, 10 ml of nutrient medium, 10 g of mud oil and 0.5 ml of mother surfactant solution (10% aqueous) were placed in a 250 ml Erlenmeyer flask. The contents of the flask were inoculated with 0.6 ml of actively growing culture from a cyclone fermentor and incubated on a rotary shaker (200 rpm) for 14 days at 25 ° C. The residual TPH content was determined after extraction with hexane. The results are given in Table 3.

Table 3 Surfactant TPH degradation (%) None 46 lgepalM R CO-630 66 BiosoftMR EN-600 63 SorbaxMR PM030 42 WitcomulMR 4078 41 Marlipal R O 1 3/120 45 All the surfactants gave an oil-in-water emulsion. Of the 5 surfactants tested, 2 surfactants viz. Igepal CO-630 and Biosoft EN600 were more effective. Approximately 66% degradation of TPH was achieved in the presence of Igepal's surfactant, compared to 46% in a control run in the absence of any surfactant.

EXAMPLE IV The effect of sludge concentration on TPH biodegradation was investigated using two different slurries at concentrations of 20%, 50% > and 90%. Each set of flasks contained the following: (a) 16 ml of nutrient medium and 4 g of mud; (b) 10 ml of nutrient medium and 1 g of mud; (c) 20 g of mud and 2 ml of nutrient medium of strength 1 0x. The flasks (250 ml) were inoculated with 600 ml of actively growing culture from a cyclone fermentor, and incubated on a rotary shaker (200 rpm) at 25 ° C for 14 days.

The results are given in Table 4.

Table 4 Mud type Mud concentration TPH degradation (% v / v start amount) Mud A (a) 20 70 (b) 50 56 (c) 90 36 Mud B (a) 20 91 (b) 50 81 (c) 90 56 It was found that the concentration of sludge affected the degree of TPH degradation.

EXAMPLE V A medium referred to herein was formed as an NPK medium when replacing, KH2PO4 and Na2HPO4, in the nutrient medium, with NPK (nitrogen: phosphorus: potassium) fertilizer (1 5:30: 15) at a proportion of 0.8 g / l. all other components in the medium were the same as described above. The experiments were conducted with two different muds. The Erlenmeyer flasks contained 50% v / v of NPK medium and 50% v / v of sludge together with 0.25% of surfactant (Igepal CO-630) based on the total culture volume.

Other conditions were the same as those described in Example ll l. The results are given in Table 5.

Table 5 Mud source Half TPH degradation (% start amount) Mold To Nutrient 60 NPK 58 Mud B Nutrient 73 NPK 71 No significant difference was observed between the results obtained with basal medium and NPK medium.

EXAMPLE VI The biodegradation of TPH was carried out in different sludges in flasks under agitation conditions. Erlenmeyer flasks containing NPK medium and mud (50:50, v / v) were inoculated with the mixed culture actively growing, and incubated for 14 days at 30 ° C.

Table 6 Mud type Mud concentration TPH degradation (%) (%) A 50 61 B 50 76 D 12.5 54 E 50 89 G 1 0 42 The results indicate that 42 to 89% degradation of TPH can be obtained using this process. The mud G, being a heavy oil mud, was the one that degraded the least.

EXAMPLE VII Alternative complex nitrogen sources were tested to yeast extract using Sludge A and Sludge B. This experiment was carried out using 50% v / v NPK medium, 50% v / v slurry and 0.25% v / v of Igepal CO-630 in 250 ml Erlenmeyer flasks incubated at 25 ° C for 14 days on a rotary shaker (200 rpm). The results are given in Table 7.

Table 7 Sludge type Complex nitrogen source Degradation of TPH (% start amount) Mud A Yeast Extract 59 Corn Infusion Solids 52 Cotton Seed Protein 51 Potato Protein 49 Mud B Yeast Extract 75 Corn Infusion Solids 85 Cotton Seed Protein 83 Potato Protein 79 All proven alternative nitrogen sources, at a final culture concentration of 0.5 g / l, gave a yield similar to 0.5 g / l of yeast extract.

EXAMPLE VIII The biodegradation of different hydrocarbon fractions was tested using Erlenmeyer flasks of mud B containing 50% v / v of sludge, 50% of NPK medium and 0.25% of Igepal CO630. After inoculation with an actively growing culture, the flasks were incubated on a rotary shaker for 14 days at 30 ° C. The entire contents of the flask was extracted once with hexane followed by dichloromethane. After centrifugation both extracts were combined and the solvent was evaporated. The residual hydrocarbon was dissolved in hexane and centrifuged. A known weight of hexane-soluble portion was passed through a column (0.75 x 27 cm) of silica gel (activated at 100 ° C overnight). Successive applications of hexane (120 ml), dichloromethane (30 ml) and chloroform: methanol (1: 1, 15 ml) produced fractions containing saturated, aromatic, and polar hydrocarbons (resins), respectively. The results are given in Table 8.

Table 8 Fraction% of hydrocarbons% of total degradation Saturated 73-77 73-77 Aromatics 1 1 -13 65-69 Resins 8-10 61 -63 The results indicate that all major components of TPH were degraded.

EXAMPLE IX This experiment was conducted to determine whether pretreatment with an advanced oxidative process (Fenton viz. H2O2 + FeSO4 reagent) could intensify the degradation of TPH in sludge. The pretreatment and subsequent biodegradation was performed in the same flask. For the pre-treatment, Sludge A was diluted with water to obtain 20 ml of 50% v / v mud concentration. The pH of the mixture was adjusted to 4.0 by adding 4N HCl. H2O2 and FeSO4 were added at concentrations of 0.3% v / v and 10 millimolar, respectively. The flasks were kept on a rotary shaker (200 rpm) at 25 ° C for 2 days. Subsequently, 2 ml of NPK medium (10-fold concentrate) were added in solid form and the pH was adjusted to 7.0 by the addition of 2N NaOH solution. The flasks were inoculated with an actively growing inoculum (600 ml) from a cyclone fermentor and incubated on a rotary shaker for a period of 28 days. The following treatments were tested: (a) no pre-treatment or surfactant addition; (b) pretreatment of Fenton reagent without surfactant; (c) addition of 0.25% of Igepal CO-630, without Fenton pre-treatment; and (d) pretreatment of Fenton reagent in the presence of 0.25%) of Igepal CO-630. The results are given in Table 9.

Table 9 Treatment Incubation time (days) 7 14 21 28% degradation of TPH None 28 42 46 53 Fenton pretreatment (48 h) 36 61 64 65 Surfactant (0.25%) 42 61 66 72 Pretreatment of Fenton (48 h) in 43 64 68 72 the presence of surfactant (0.25%) The results indicate that the pre-treatment of sludge with an oxidative agent or addition of surfactant significantly increased the degree of degradation of TPH in mud oil .

EXAMPLE X The performance of TPH biodegradation in different sludges in different types of reactors was evaluated. The reactors tested were of different configuration and scale. The biodegradation tests in Erlenmeyer flasks were carried out as described in other examples. The cyclone fermentors were as described above. The air lift reactors were fitted with sprinklers and connected to an air source. Mixing in the reactors was achieved by supplying air at a rate of 0.5 volume / minute and 0.125%) of surfactant. NPK medium and mud (50:50 v / v) were used in these experiments. All reactors were inoculated with a mixed culture actively growing. The results are presented in Table 10.

Table 1 0 Type Reactor type Scala de Ti emp o de Biodegradación de p? 'incubation of TPH sludge (liters) (days) (%) E Erlenmeyer flask 0.25 20 74 E Air lift 150 14 70 F Air lift 150 14 84 B Erlenmeyer flask 0.25 14 81 B Cyclone 1 8 85 C Air lift 18000 1 1 84 The results show that efficient sludge degradation occurs in different types of aerated reactors and at different operating scales, varying from laboratory to production scale.

EXAMPLE XI The biodegradation of TPHs in clay fines in shake flask cultures was evaluated. The flasks containing clay fines (TPH, 1 0.5% >, w / v, and medium N PK 50:50, w / v) and 0.25%, w / v of Igopal CO-630 were inoculated with an actively growing culture and incubated for 14 days at 30 ° C. The residual TPH content was determined and the results are shown in Table 1 1.

Table 1 1 Incubation time (days)% degradation of TPH 7 77 14 92 The results indicate that 92% clay fines can be achieved in 14 days using this process.

Claims (38)

1. REIVI NDICATIONS 1 . A method for the biodegradation of an oil-based sludge, said oil-based sludge comprising a mixture of petroleum hydrocarbons, said method comprising the steps of: (a) forming an aqueous solution in an oil-in-water emulsion reactor , bacterial culture and nutrients for said bacterial culture, said emulsion being oil-in-water an emulsion of said sludge based on oil in water, said bacterial culture having the capacity to grow in petroleum hydrocarbons as a single source of carbon and having been isolated of a soil contaminated with hydrocarbon or sludge containing hydrocarbon or other environments rich in hydrocarbon degrading bacteria, by means of microbial enrichment techniques using hydrocarbons in the selection medium, said reactor containing up to 50% by volume of total petroleum hydrocarbons; (b) maintaining said aqueous solution under aerobic conditions in the reactor at a temperature of at least 10 ° C for a period sufficient to reduce the amount of hydrocarbons by at least 25%, and at a favorable pH for the promotion of bacterial growth and degradation of hydrocarbons; and (c) discharging the aqueous solution having a reduced amount of total petroleum hydrocarbons from said reactor.
2. 2. The method of claim 1, wherein said nutrients comprise chemical components of bacterial cells in proportions corresponding to relative proportions in naturally occurring bacterial cells, and delivered at concentrations that promote high levels of bacterial growth and high proportions of bacteria. degradation of hydrocarbons.
3. 3. The method of claim 1 or claim 2, wherein said nutrients comprise bioavailable nitrogen and phosphorus.
4. 4. The method of any one of claims 1 -3, wherein the reactor contains about 5-50% by volume of total petroleum hydrocarbons.
5. The method of claim 4, wherein the reactor contains about 10-30% by volume of total petroleum hydrocarbons.
6. The method of any of claims 1-5, wherein said petroleum-based sludge contains extractable hydrocarbons with hexane in an amount in the range of up to 500,000 ppm.
7. The method of claim 6, wherein the amount of extractable hydrocarbons with hexane is in the range of 65,000-250,000 ppm.
8. The method of any of claims 1-7, wherein the petroleum hydrocarbons consist of mixtures of saturated hydrocarbons, aromatic hydrocarbons, hydrocarbon resins and asphaltenes.
9. 9. The method of any of claims 1-8, wherein the amount of nitrogen is in the range of 50-1000 ppm and the amount of phosphate is in the range of 10-200 ppm.
10. The method of any one of claims 1-9, wherein the nitrogen compound is an ammonium ion, nitrate or organic nitrogen, and phosphorus is phosphate. eleven .
11. The method of any of claims 1-10, wherein there is a nonionic surfactant in an amount sufficient to form said oil-in-water emulsion.
12. The method of any of claims 1-10, wherein there is an anionic surfactant in an amount sufficient to form said oil-in-water emulsion.
13. The method of claim 1 or claim 12, wherein the amount of surfactant is less than 2500 ppm.
14. The method of claim 1 or claim 12, wherein the amount of surfactant is less than 1500 ppm.
15. The method of any of claims 1-14, wherein the nutrient contains at least one of magnesium, manganese, sulfate, organic sulfur, calcium, ferric ion, copper.
16. 16. The method of claim 1, wherein the nutrient additionally contains cobalt, zinc, boron or molybdenum.
17. The method of any of claims 1-16, wherein said aqueous solution contains a surfactant, the ratio of the amount of petroleum hydrocarbon to surfactant being at least 40: 1.
18. 18. The method of any of claims 1-17, wherein the aqueous solution is maintained in step (b) in at least two reactors in series before being discharged in step (c).
19. 19. The method of any of claims 1-18, wherein the aqueous solution is maintained in step (b) for a holding time of at least 7 days.
20. The method of any of claims 1-19, wherein the relative composition of the nutrients reflects the known relative composition of components required for the growth of bacteria. twenty-one .
21. The method of any of claims 1-20, wherein a proportion of the degraded sludge in the reactor is retained after discharge as an inoculum for the next batch of sludge.
22. 22. The method of any of claims 1-21, wherein the reactor is operated as a batch-fed, continuous or semi-continuous system.
23. 23. The method of any of claims 1-22, wherein the sludge is pre-treated chemically or physically to improve biodegradability prior to, or during biodegradation.
24. The method of any one of claims 1-23 in which the degradation of partial petroleum hydrocarbons occurs resulting in the separation of an aqueous and an oily phase, said oily phase being recycled to the next batch of sludge degradation.
25. 25. The method of claim 24, wherein the separated oil phase is recovered.
26. 26. The method of claim 24, wherein the separated aqueous phase is rich in hydrocarbon degrading bacteria and used as a bacterial inoculum to accelerate bioremediation of soil contaminated with hydrocarbons.
27. 27. The method of claim 24, wherein the bacteria are recovered from the aqueous phase for subsequent use as a bacterial inoculum.
28. 28. The method of any of claims 1-27, wherein the sludge is a byproduct of coal processing.
29. 29. The method of any of claims 1 -27, wherein the sludge is an oil refinery sludge.
30. 30. The method of any one of claims 1-27, wherein the slurry is in the form of oil fines containing clay.
31. 31 The method of any of claims 1-27, wherein the iodine is obtained from the bottom of an oil storage tank.
32. 32. The method of any of claims 1-27, wherein the sludge is an oil residue from a wellhead on the ground or from the washing of a hold on a tanker.
33. 33. The method of any of claims 1-27, wherein the petroleum residue comprises treating emulsions or sludge oil.
34. 34. A method for the biodegradation of an oil-based sludge, said oil-based sludge comprising a mixture of petroleum hydrocarbons, said method comprising the steps of: (a) forming an aqueous solution in an oil-in-water emulsion reactor , bacterial culture and nutrients for said bacterial culture, said emulsion being oil-in-water an emulsion of said sludge based on oil in water, said bacterial culture having the capacity to grow in petroleum hydrocarbons as a single source of carbon and being native bacteria in the oil-based mud, such native bacteria multiplying and degrading the mud, said reactor containing up to 50% by volume of total petroleum hydrocarbons; (b) maintaining said aqueous solution under aerobic conditions in the reactor at a temperature of at least 10 ° C for a period sufficient to reduce the amount of hydrocarbons by at least 25%, and at a favorable pH for the promotion of bacterial growth and degradation of hydrocarbons; and (c) discharging the aqueous solution having a reduced amount of said hydrocarbons from said reactor.
35. 35. The method of any of claims 1-34, wherein the inoculum comprises one or more microbial degrading hydrocarbon strains produced by fermentation under non-sterile conditions.
36. 36. The method of any of claims 1 -33, wherein the temperature in step (b) is in the range of 20-37 ° C.
37. 37. The method of any of claims 1 -33, wherein the temperature in step (b) is in the range of 27-33 ° C.
38. 38. The method of any of claims 1-27, wherein the petroleum-based mud is mixed with soil. SUMMARY A method for the biodegradation of an oil-based sludge comprising a mixture of petroleum hydrocarbons is described. The method comprises forming an aqueous solution in an oil-in-water emulsion reactor of petroleum-based mud, bacterial culture and nutrients for bacterial culture, the bacterial culture having the ability to grow in petroleum hydrocarbons as the sole source of carbon and having been isolated from a soil contaminated with hydrocarbons or mud containing hydrocarbons or other environments rich in hydrocarbon degrading bacteria, keeping the aqueous solution under aerobic conditions in the reactor at a temperature of at least 10 ° C for a sufficient period to reduce the amount of hydrocarbons by at least 25%, and discharging the aqueous solution having a reduced amount of hydrocarbons from the reactor. The method can be used in mud containing aromatics, resins and asphaltenes.