DE4340808C1 - Method for disposing of polycyclic aromatic compounds from contaminated particulate materials by biodegradation - Google Patents

Abstract

A process for disposing of polycyclic aromatic compounds from contaminated particulate materials by biodegradation is described, in which the materials are contacted with an aqueous solution of methylpurines and the biodegradation of the polycyclic aromatic compounds is effected by the microorganisms contained in the particulate materials and/or by addition of microorganisms capable of biodegradation. According to a preferred embodiment, caffeine is used as methylpurine and bacteria and/or moulds are used as microorganisms.

Description

The present invention relates generally to microbial Remediation or decontamination of particulate materials such as Soils, sediments, sewage sludge and filter dust, which with polycy aromatic compounds are loaded. Especially The present invention relates to a method for soil sane tion using methylpurines and microorganisms are able to remove polycyclic aromatic compounds to build.

It is known that a variety of renovation needs particulate materials especially in densely industrialized areas with a high content of polycyclic aromatic Have connections (PAV). The impurities are particularly especially due to contaminated landfills or Production plants (gas works, coking plants, oil refineries, tar fa briken, wood impregnation plants) as well as by transport accidents and damaged pipes. The concentrations often exceed the PAV pollutants mentioned in recommendations and guidelines tolerance values many times over.  

For the purposes of the present invention, the "polycyclic aromatic compounds (PAV) "both polycyclic aromati hydrocarbons (PAHs) as well as azaarenes, thiaarenes, Oxaarene and halogen-containing aromatic compounds and their Methyl and hydroxyl derivatives.

While representatives of the PAV group with three or fewer con densified rings by different types of bacteria as the only ones Carbon and energy source can be completely degraded the higher polycyclic aromatic compounds conditions with more than three condensed rings to the difficult degradation possible soil contaminants, whereby their degradability with stei gender The number of condensed rings decreases. However, it is knows that it is these higher condensed PAV that Chen have carcinogenic potency. Over the past few years extensive studies have shown that various microorganisms such as bacteria and fungi are able to also to decompose higher condensed PAV cometabolically.

About the metabolism and biodegradation of polycycli aromatic hydrocarbons from bacteria and fungi has been reported several times (C.E. Cerniglia, Microbial transforma tion of aromatic hydrocarbons, petroleum microbiology (R.M. Atlas, Her.), Pp. 99-128, Macmillian, New York (1984b); C.E. Cerniglia, Microbial metabolism of polycyclic aromatic hydrocar bons, Adv. Appl. Microbiol., 30, pp. 31-71, (1984c); C.E. Cerni glia and M.A. Heitkamp, Microbial degradation of polycyclic aromatic hydrocarbons (PAH) in the aquatic environment, Metabo lism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environments ment, (U. Varanasi, Her.) 5. 41-68, (1989b), CRC Press, Boca Raton).

Tables 1 and 2 below are the metabolic processes of fungi and bacteria in the breakdown of polycyclic aroma table hydrocarbons listed as examples.  

Table 1: PAH metabolic processes in mushrooms

Table 2: Metabolism of polycyclic aromatic hydrocarbons in bacteria *

In the eukaryotic organisms, aromatic double bin to form arene epoxides, which can be generated in vivo either by spontaneous isomerization to phenols or enzymatically by Hy Drasen converted to trans-Diyhdrodiolen with water become. In addition, mostly in minor quantities also quinones and in the case of naphthalene also tetralones as Metabolites detected.

Trans-dihydrodiols can again be used as substrates for monooxygena serve and are converted to trans-dihydrodiol epoxides, which in particular in connection with the development of cancer higher animals and humans play a special role. By Water attachment to the diolepoxides becomes the non-toxic Tetrahydrotetrole formed together with the above Metabolic products are referred to as phase I metabolites. They are detoxified by conjugation with sulfuric acid, Glucuronic acid, sugars or glutathione to sulfates, glucuro niden, glucosides or glutathionates, the so-called Pha se II metabolites.

The individual steps of this metabolism that have been demonstrated in mushrooms Sel processes are listed in Table 1. A special position take the white rot fungi (Phanerochaete chrysosporium, Trametes versicolor), in which both monooxygenases and lig ninperoxidase (s) are involved in the metabolism of PAHs (J.B. Sutherland, A.L. Selby, J.P. Freeman, F.E. Evans, C.E. Cerniglia, Metabolism of phenanthrene by Phanerochaete chryso sporium, appl. Environ. Microbiol., 57, pp. 3310-3316, (1991)). In addition to phenols and trans-dihydrodiols, various PAHs such as anthracene, pyrene, benz [a] anthracene, perylene and benzo- [a] pyrene also detected quinones as phase I metabolites (K.E. Hammel, B. Kalyanaraman and T.K. Kirk, Oxidation of polycyclic aromatic hydrocarbons and dibenzo (p] dioxins by Phanerochaete chrysosporium ligninase, J. Biol. Chem., 261, pp. 16948-16952, (1986)).  

In prokaryotes, on the other hand, the metabolism of lower PAHs catalyzed by dioxigenases and leads primarily to cis-diyhdro diols, from which phenols can be formed by elimination of water nen. The further metabolism leads to too low after opening the ring molecular connections and finally to complete mine ralization of the substrate. The metabolic performance varies whose bacteria are summarized in Table 2.

To the conventional methods for the renovation of z. B. PAV- polluted floors include thermal processes and washing proceed the on-site, in-situ and fermentative processes taking advantage of the given soil microflora, which is made up of assemble a variety of different bacteria and fungi can. In the last three methods mentioned, Inten Activation of the microbial degradation additionally adapted mixing cultures are used.

In the on-site procedures, the soil to be treated is excavated ben, mixed with straw or bark schnitzel and as with the Composting in rents or rotary drum reactors ventilated. To the Dismantling of xenobiotic compounds can also result in soil excavation Lich with white rot fungi such as Phanerochaete chry sosporium can be inoculated, which are capable of different Environmental pollutants such as DDT, PCB, dioxins and lindane up to one break down oxidatively to a certain degree.

The in situ process uses nutrients, surfactants and adap microorganisms directly incorporated into the soil. Contamination in deeper soil layers can be caused by pumping around pen of a mixture of water, nutrients and microorganisms between different swallowing and production wells microbial be dismantled. As chemical oxygen donors are nitrates and hydrogen peroxide suitable in aqueous solutions in the Soil. In situ remediation with chemical However, oxygen donors require a careful process control to damage the soil microflora through over  Avoid dosing the oxygen dispenser. The choice of Appropriate process is based on the soil structure and depth location depending on the contamination. For example, for Ver impurities in deep layers of soil only microbial le in situ method applicable.

In the fermentative process, the soil is excavated in transferred a container suitable as a fermenter (bioreactor) and incubated.

The increase in microbial degradation performance in the previous described renovation process was on the one hand by Opti oxygenation and nutrients tion and on the other hand by adding selected metabolism specialist or mixed populations reached in the Laboratory test especially when using fermentative ver promising re in a suitable bioreactor results with regard to the degradation of difficult to metabolize con taminants had rendered. Especially the biotechnological sania However, soil with high levels of PAV has a number of serious problems uncovered.

Because of the diverse interrelationships between abioti and biotic factors of a biotechnological sania the entire condensers could be completely dismantled based PAV have not yet been achieved. Rather, it turned out after initially clear mining a plateau, its height apparently from the bioavailability of the contaminants for the Is dependent on microorganisms.

It must be assumed that bioavailability from the "solvent availability", which is characterized by the extractant is determined and the extraction of the solubilizing particulate matrix with solvents PAV share concerns (see Examples 1 and 2); (G. Grimmer, K.-W. Naujack, D. Schneider, Profile analysis of polycyclic aromatic  hydrocarbons by glass capillary gas chromatography in atmosphe ric suspended particulate matter in the nanogram range collec ting 10 m³ of air, Fres. Z. Anal. Chem., 311, pp. 475-484, (1982)).

Various attempts have been made to increase bioavailability condensed PAV in the soil through surfactants and solubilizers (K. Mackenbrock, The R&D cooperation model of the Deutsche Babcock Anlagen AG in the development of a biologi process for the renovation of coke oven floors, seminar "Biolo Soil remediation: on the way to market-oriented processes ren? ", MülheimlRuhr, 5.12.1989, 6S., (1989)). These experiments have proven to be unsuccessful.

The object of the present invention is therefore to provide a superior method for removing PAV from contami particulate materials.

To solve the problem, the method according to the main claim proposed, with specific embodiments of the invention are set out in the subclaims.

It has surprisingly been found that the inventive Procedure for the microbial remediation of contaminated particulate Materials leads to excellent results, in particular it is possible according to the invention to determine the bioavailability of the con taminants significantly increase and thus a more extensive To achieve degradation of the higher-condensed PAV than previously was possible.

In the method according to the invention, those with polycycli aromatic compounds contaminated particulate Materials cleaned by biodegradation by man (a) the materials with an aqueous solution of methylpu brings rine into contact and (b) the biodegradation of the poly cyclic aromatic compounds by means of the in the particulate  Lärische materials contained microorganisms and / or by Addition of microorganisms capable of biodegradation instead can be found.

Methylpurines preferred according to the invention are selected from the group consisting of theophylline, theobromine and caffeine selected, with caffeine being particularly preferred. The methylpurine is in Form of an aqueous solution, preferably an aqueous nutrient solution such as B. a 2 wt .-% biomalt nutrient solution sets, the nutrient medium or nutrient solution with regard to the microorganisms to be used selected and opti can be lubricated. According to the invention, it is preferred that the Me thylpurine in the aqueous solution in a concentration of 0.01 to 1 wt .-%, with a concentration of 0.1 to 0.5 % By weight is preferred. According to a particularly preferred embodiment The aqueous solution used according to the invention has a shape the methyl purine in a concentration of 0.3% by weight.

The solution containing the methylpurines can be treated sprayed on the ground, mixed with it, or into one Fermenter introduced and in this way with the contaminated Be brought into contact with the ground; the procedure can be used for the known on site, in situ and fermentative processes in Framework of their usual procedure.

According to the invention, it is particularly in use tative process preferred, the solution before applying Mi add croorganisms that are able to break down PAV.

The particulate suitable according to the invention for decontamination Materials include soils, sediments, sewage sludge and filters dust.

According to a preferred embodiment of the invention The process leaves the biodegradation in step (b) under Addition of fungi and / or bacteria take place.  

Suitable bacteria include the genera Pseudomonas, Alcali aenes, Arthrobacter, Micrococcus, Vibrio, Brevebacterium, Cory nebacterium, Beÿerinckia, Aeromonas, Mycobacterium and Strepto myces. Particularly preferred representatives of the genus Pseudomonas include the species Pseudomonas putida and Pseudomonas vesicu laris. Preferred representatives of the genus Alcaligenes include the Species Alcaligenes faecalis and Alcaligenes denitrificans. In the species Streptomyces becomes the species Streptomyces flavovi rens preferred.

According to the invention, the mushrooms comprise the preferred genera Cunninghamella, Aspergillus, Neurospora, Mucor, Pleurotus, Tri choderma, Fusarium, Phanerochaete, Sporobolomyces, Saccharomy ces, candida and rhodotorula, of which the genera Mucor, Cunninghamella, Trichoderma, Fusarium, Pleurotus, and Phanero chaete are particularly preferred. From the genus Cunninghamel la are preferably the species Cunninghamella bainieri and Cunninghamella elegans includes. The preferred species of the Gat Aspergillus, Neurospora and Mucor are the species Asper gillus ochraceus, Neurospora crassa and Mucor circinelloides. The species are preferred representatives of the other genera Pleurotus ostreatus, Trichoderma viride, Fusarium oxysporum, Phanerochaete chrysosporium, Saccharomyces cerevisiae and Can dida utilis.

According to the invention, the bacteria and / or fungi are the above mentioned genera or species individually or in combination used and preferably immobilized before their addition, wherein as carriers all common carriers and porous materials like in particular glass beads, expanded clay, Siran carriers and metal wire and plastic film.

As explained above, the method according to the invention can be carried out in one Fermenter or as part of a conventional on-site or in situ method are used.  

According to a preferred embodiment of the invention, the Soil contaminated with PAV and depending on the Condition and composition if necessary sieved, ver grind or otherwise homogenized, and in a ferment introduced. A nutrient solution is then added solution, for example 0.3% by weight of caffeine, 2% by weight of organic salt and can contain 10³ to 10¹⁰ spores or cells per ml and de volume depending on the nature of the soil and the ones to be used Microorganisms depends, the volume ratio between Liquid and soil, for example, be 1:10 to 100: 1 can.

The individual conditions of the fermentative process as ins special temperature, duration and nutrient supply are of the nature, composition and degree of contamination of the Soil and the respective optimal cultural conditions of the microorganisms to be used are dependent on the subject man in the field of biotechnological process control easy to choose, the temperature usually between 20 and 42 ° C, but in individual cases also higher or lower can be, provided the temperature optimum of the Mi microorganisms require a different temperature or through which Other temperatures can be specified.

Furthermore, the nutrient solution or proposed according to the invention if necessary, individual components of the same for acceleration of the restructuring process continuously or successively in the Fermenters are initiated.

The progress of microbial degradation contained in the soil The polycyclic aromatic compounds can be regulated moderate sampling and z. B. gas chromatographic analysis of remaining pollutant levels are monitored. The renovation The restoration process will be terminated if the desired renovation goal is achieved.  

The method proposed according to the invention is the conventional one process in particular in two main areas Think about points. On the one hand, the microbial breakdown of PAV by adding methyl purines such as caffeine accelerate, on the other hand, the treated according to the invention Materials compared to otherwise in the same way but without Methylpurine treated materials significantly decreased rest stop at PAV.

The invention is further illustrated by the following examples explained.  

example 1

To compare the extractability of polycyclic aromati connections from a contaminated floor of a coking plant Water or an aqueous 1% by weight caffeine solution carried out the following experiment:

In each case 10 g of one loaded with 600 mg benzo [a] pyrene / kg Rilized soil was placed in 2 Erlenmeyer flasks, the 100 ml of water or 100 ml of a 1 wt .-% caffeine solution contained The contents of the flask were mixed thoroughly and under Stir heated at 100 ° C for one hour. After the ex traction, the amount of liquid was decanted and the content determined on benzo [a] pyrene in the liquid medium. This process was repeated three times, using the two approaches Percentage of benzo [a] pyrene in the liquid medium 0.1 to 0.5% was and gradually decreased. Then followed extraction with toluene, through which 99% of the pollutant from the Soil could be brought into solution. This result shows that the extractable portion of water as a solvent Not harmful by the use of caffeine alone being affected.

Example 2

To determine the effect of longer downtimes on the Ex traceability of various polycyclic aromatic Connections from a contaminated, sterilized floor was made the experimental setup according to example 1 is carried out with the deviation chung that the individual batches each have an aqueous 0.3 wt. % caffeine solution included for 15 days at room temperature were incubated. After the extraction time he followed each time after decanting the caffeine solution used a subsequent extraction with boiling toluene for a period of  1 hour. Then the concentrations of the PAV in the both extraction liquids measured. The results are in summarized in Table 3 below.

Table 3: Extractability of PAV from a PAV-contaminated soil with caffeine-containing water (0.3% by weight) in 15 days at 20 ° C (mg / kg)

These results also show that extraction with caffeine only a small extract under ambient conditions tion of the pollutants leads.

Example 3

The quantitative evaluation of the method according to the invention using methylpurines and microorganisms in the frame A fermentative process was carried out with a sterilizer th, sandy Heideboden, which with different PAV connection was burdened.

The following series of experiments was carried out in triplicate and after different downtimes on the residual PAV is looking for.

In each case 2 g of the contaminated heath land were in three Erlenmey flask given, each 50 ml of a biomalt nutrient solution contained, which neither caffeine nor microorganisms (unhindered deltered soil), only microorganisms (treated soil without Complexing agent) or caffeine and microorganisms (treated ter soil with complexing agent) included. As a microorganism was the mushroom Mucor circinelloides IMBMC 1.91, which previously made an industrial floor, in a concentration of 10⁵ spores per ml used. As methylpurine, caffeine was found in a concentration of 0.3 wt .-% used. The approaches were incubated as a shake culture and after every 15, 30 and 45 Days evaluated.

The results of this experiment are in Tables 4 to 6 reproduced.  

Table 4: Comparison of the degradation performance of Mucor circinelloides with and without PAV complexing agent after 15 days using the example of a PAV-contaminated sandy heathland (PAV mg / kg; double values)

As can be seen in Table 4, the results of the Repeat the experiment well with and without the complexing agent caffeine bar (double approach with small deviation) and zei significant differences in the mining performance of the used zen microorganism with regard to the presence in the soil sample pollutants. While in the presence of caffeine already after 15 days a significant decrease in the PAV in the ground The same double sample can be found without pollutants  Add the complexing agent under the same conditions only in Range of more soluble PAV such as fluoranthene and pyrene recognize a measurable decrease. The degradation performance of the mushroom shows with the addition of the complexing agent caffeine breakdown rates of 64% and 67% respectively, the degradation rate in the case of benzo [a] pyrene was approximately 40% after 15 days of incubation. The results Nisse of the following tables 5 and 6 show that the invent effect according to the invention with a correspondingly longer incubation period (30 or 45 days) could be increased as expected.

Table 5: Comparison of the degradation performance of Mucor circinelloides with and without PAV complexing agent after 30 days using the example of a PAV-contaminated sandy heathland (PAV mg / kg; double values)

Table 6: Comparison of the degradation performance of Mucor circinelloides with and without PAV complexing agent after 45 days using the example of a PAV-contaminated sandy heathland (PAV mg / kg; double values)

The results of Table 6 show that the set by the use of caffeine according to the invention Let effect intensify even further. For example, Ben zo [a] pyrene degraded to 86.5% when caffeine was added, while in Comparative approach without caffeine only a degradation rate of 49.7% was aimed.

In the graphic below, they are made using the inventions Summary of the method determined results shown.

In another experiment, the Mucor mushroom became circinelloides IMBMC 1.91 mounted on Siran carriers and among the previously be conditions of a conventional shake culture 15 Days with those listed in Table 7 below Pollutants with or without the addition of 0.3% by weight of caffeine beer. The results shown clearly show that by Process proposed according to the invention achieved effect.

Table 7: Comparison of the degradation performance of immobilized Mucor circinelloides with and without PAV complexing agent after 15 days using the example of a PAV-contaminated sandy heathland (PAV mg / kg; double values)

Claims (12)

1. A process for eliminating polycyclic aromatic compounds from contaminated particulate materials by biological degradation processes, characterized in that

• a) bringing the materials into contact with an aqueous solution of methylpuri and
• b) the biodegradation of the polycyclic aromatic compounds takes place by means of the microorganisms contained in the particulate materials and / or by addition of microorganisms capable of biodegradation.

2. The method according to claim 1, characterized in that Methylpurine from the group consisting of theophylline, Theo bromin and caffeine can be used. 3. The method according to claim 2, characterized in that as Methylpurine caffeine is used. 4. The method according to any one of the preceding claims, characterized characterized in that the methylpurine in aqueous solution in a concentration of 0.01 to 1 wt .-% is used. 5. The method according to claim 4, characterized in that Me thylpurine in a concentration of 0.1 to 0.5% by weight is used. 6. The method according to claim 5, characterized in that Me thylpurine used in a concentration of 0.3 wt .-% becomes.   7. The method according to claims 1 to 6, characterized in net that soils, sediments, clarifiers as particulate materials sludge and filter dusts are treated. 8. The method according to claims 1 to 7, characterized in net that the biodegradation in stage (b) under Zu administration of fungi and / or bacteria takes place. 9. The method according to claim 8, characterized in that one Mushrooms of the genera Mucor, Cunninghamella, Trichoderma, Fusarium, Pleurotus and / or Phanerochaete used. 10. The method according to claim 9, characterized in that as Microorganisms Mucor circinelloides, Cunningha mella bainieri, Cunninghamella elegans, Trichoderma viride, Fusarium oxysporum, Pleurotus ostreatus and / or Phanero chaete chrysosporium can be used. 11. The method according to claims 8 to 16, characterized in net that the fungi and / or bacteria are immobilized on a carrier material used. 12. The method according to claims 1 to 11, characterized in net that you can biodegradation in a fermenter or as part of a conventional in situ or on site process rens takes place.