CZ2010908A3 - Base material for formation of biofilm - Google Patents

Abstract

Biofilm-forming material in packed biological permeable reactive barriers, for the remediation of groundwater with polluted organic compounds, contains 5-6 parts by volume of crushed PET size fractions of 3 to 5 cm, 3 to 2 parts by volume of expanded clay - expanded clay fraction 0.8 to 1, 6 cm and 2 parts by volume of the fraction 0.5 to 1 cm, while the crushed PET and clay are mixed together mechanically, the gravel being then added as the top / front and bottom / back layers of the filling, depending on the direction of groundwater flow.

Description

Carrier material for biofilm formation

Technical field

BACKGROUND OF THE INVENTION The present invention relates to a carrier material for biofilm formation in packed biological permeable barriers for the remediation of groundwater contaminated with organic compounds.

BACKGROUND OF THE INVENTION

In recent years, the fifth trend is to use innovative remediation technologies to treat contaminated groundwater. The main reason for deviating from classical remediation methods is their lower efficiency and the associated higher time and cost. Significant development of permeable reactive barrier technology (PRB) is then determined primarily by the success of the first real applications and also by the advantage of the in-situ application where the remediation takes place directly in the rock environment without the need to pump contaminated groundwater.

A permeable reactive barrier is generally defined as an in situ zone formed by a reactive material through which contaminated groundwater flows in the natural mode. The reactive medium placed in the barrier decomposes, sorbs, precipitates or otherwise removes organic substances, metals or radionuclides. Thus, with regard to the type of contamination to be cleaned, the barriers may contain solid reactive materials or liquid reagents for the decomposition of organic matter and the immobilization of metals, or also nutrients or oxygen for assisted in situ biodegradation.

PRBs carried out to date have been applied primarily to remove halogenated aliphatic hydrocarbons, in particular chlorinated ethylenes, or heavy metals, in particular Cr ⁶ ', by chemical reduction on a medium formed by zero-valent iron, commonly abbreviated as ZVI, based on the term "zero valeni iron". ". Only a small part of the reactive barriers were built to remove other types of pollution, such as oil, aromatics or benzene. In the reactive barrier technology, these organic substances are usually removed by sorption on activated carbon, hereinafter referred to as AU, or by natural attenuation supported by the supply of oxygen to the reactor. Oxygen is usually supplied to the groundwater by so-called air-sparging barriers, which consist of one or more lines of injection wells built across the groundwater flow direction. These bores are forced into the saturated zone

F t l F <1 1 f 4 4 <

-2 atmospheric oxygen as an electron acceptor, mineral nutrients are supplied as a rule to ensure an optimum ratio of nitrogen, N and phosphorus, hereinafter P.

A relatively new issue in the field of application of biological PRBs is the use of so-called packed biological barriers. In this case, the permeable barrier reactor is filled with a solid material on which a bio film of degrading organisms is produced, whether autochthonous, naturally occurring at the remediated site, or alochtonous, artificially cultured in the laboratory and then delivered to the wall reactor. At the same time, nutrients N and P can also be supplied in this case, usually in the form of fertilizers. The application of barriers of this type can be documented using an example of a so-called denitrification barrier filled with organic carbon revived by Pseudomonas sp.

It follows from the above that the principle of utilizing packed biological barriers is already known in the prior art and is also mentioned in several European and worldwide patents. The patent claims of these solutions are based on a different material type of the filler, the carrier of the bio film, the genus, or the like. the type of microorganism used, bacteria, yeast, forming the degradation layer of the bio film on the filling and the technical method of reactor configuration, possibly also the whole system of biological PRB.

in

For example, International Patent Publication WO2004080620A2 discloses the use of inert materials such as pumice or expanded clay, which is a baked cellular clay sold under the trade name keramzite, as a carrier of a bio film consisting of the anaerobic bacterial genera Desulfotomaculum and Desulfovibrio for biotransformation of groundwater contamination. The possibility of using microorganisms capable of degrading the present contaminants and their immobilization on granular activated carbon, hereinafter referred to as GAC based on the term "granular activated carbon" in a medium of the polyvinyl alcohol type, hereinafter referred to as PVA, is disclosed in WO0132566A1. A similar principle is also described in US patent 6337019S1, which describes the use of encapsulated microorganisms in a drop of PVA with the addition of a 3% mixture of GAC and sand.

Another international patent WO9849106A1, in conjunction with US patent US6719902B1, discloses the use of ZVI for the introduction of anaerobic conditions in a rock environment to promote subsequent anaerobic biotransformations of mixed contamination by a series of enumerated autotropic bacteria in various technological arrangements including permeable reactive barrier systems. At the same time, materials such as glass, concrete, metal, zeolite, mineral, fiber, glass wool, plastic,

3

polymer or resin. Also US2007025820A1 describes the use of an addition of metals, in this case polyvalent, in combination with fibrous organic materials in the form of composites, to remove organic contamination from the rock environment. Said composites are shaped so that they can also be used in reactive barriers.

The use of a combination of power fly ash and the addition of a biologically active substance containing bacteria capable of degrading organic contamination is claimed by the International ^ patent.

IN'

WO9315831A1. Using the above combination, sorption of the contamination to the fly ash occurs in the first phase and then, in the second phase, it is biodegraded by the microorganisms present.

The use of biogenic material - a mixture of hydroxyapatite, rosaceae, bone and fishmeal as a substrate and supporting surface of microorganisms for the decontamination of ground and surface waters is disclosed in US patent US200908473 UAl. The use of grain residues and barley extracts from beer production to promote biodegradation of organically contaminated soils and groundwater is disclosed in the international patent WCX2OO6123O81A1. This document also presents the possibility of coating cereal residues with a polyol ester of C ₁₂ to C ₂₄ fatty acids, which is subsequently biodegraded when the soil is degraded.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a biofilm-forming carrier material in packed biological permeable barriers for the remediation of groundwater contaminated with organic compounds that is readily available, inexpensive, and at the same time ensures the formation of a stable degradation biofilm.

This is largely achieved by the carrier material for biofilm formation in the filling barriers according to the invention, which consists essentially in a mixture of crushed waste plastic bottles made of polyethylene terephthalate, hereinafter called PET, supplemented with a certain proportion of expanded clay (keramzitwa gravel).

The carrier material is optimal when it contains 5 to 6 parts by volume of crushed 3-5 cm size PET, 3 to 2 parts by volume of expanded clay-ceramite, 0.8-1.6 cm fraction and 2 parts by volume of 0.5 gravel -1 cm, with crushed PET and expanded clay being mechanically mixed together, and the gravel forms the top / front and bottom / back layers of the filler depending on the direction of groundwater flow.

«4 i« r * r * f · · i;

. 4 ’

For technological reasons, it seems advantageous if, for the vertical flow of purified groundwater through the barrier, the carrier material is embedded in a cassette of stabilized polypropylene, hereinafter referred to as stPP.

With respect to the recovery of the carrier material, it appears advantageous if it contains at least one degrader inoculum selected from a Gram-negative bacterial group, including Achromobacter xylosoxidans DEK 2, Citrobacter freundii DEK 1C, Comamonas acidovorans ANI 1, Cupriavidus metallidurans DEK IR, Ochrobactrum anthropi NAF B1, Pseudomonas putida 161, Pseudomonas veronii 145, V1-2 and V2-1 and Rahnella sp. DEK 1.

In order to promote the growth of the bio film on the carrier material, it is expedient if N and P are added in a weight ratio of 1: 0.6 to 1.2 in the form of a mineral fertilizer, in a concentration of 1 to 3.5 g / l.

It is advantageous with regard to the remediation efficiency when the carrier material is used for the remediation of aromatic and polyaromatic pollutants.

List of drawings

The invention will be further elucidated by the following drawings in which Figure 1 shows the protein content of test materials after completion of laboratory cultivation in the presence of phenol and the Cupriavidus metallidurans DEK IR bacterial degrader, Figure 2 shows the phenol biodegradation in the presence of selected carriers by Cupriavidus metallidurans DEK. IR, graph 3 of the growth curve of Ochrobactrum anthropi NAF B1 on various energy and carbon sources, graphs 4 and 5, the degradation efficiency curve and the overall stress characteristic of columns 1 and 2 simulating real site conditions, graph 6, degradation efficiency curve and the chamber III overall stress characteristic. pilot plant barriers in real conditions and in graph 7 contents of cultivable bacteria in groundwater collected at the inlet and outlet of chamber III. pilot barriers; Table 1 total amount of proteins on materials after cultivation, Table 3 effect of mineral content of N, P on the biodegradation of naphthalene by Ochrobactrum anthropi NAF Bl, Table 4 effect of aniline aeration rates for biodegradation of organic substances by Achromobacter xylosoxidans DEK 2 and Rahnella sp. DEK 1 and in Table 5 the half-lives of contamination in the individual biological barrier chambers; and at the same time in Figure 1 a schematic representation of a permeable reactive barrier, Figure 2 a possible 5 'barrier filling arrangement, Figure 3 a barrier filling arrangement in the case of vertical flow, and Figure 4 a schematic of aeration chamber 61 in the barrier reactor; design of pilot plant bioreactor with pre-aeration chamber 61 and in figure 7 identification of ribosomal DNA in packed chambers II. and III. reactor model barrier.

Description of exemplary embodiments

The carrier material is based on the use of a mixture of crushed plastic waste bottles made of polyethylene terephthalate, ie a thermoplastic from the polyester family, known by the abbreviation PET, supplemented by the proportion of expanded clay - clay and gravel. According to the invention, the essence of preparing 1 m ^{3 of} charge consists in combining 5 to 6 parts by volume of crushed δ

PET size fraction 3 '? 5 cm, 3 to 2 parts by volume of expanded clay - ceramitite fraction 0.8 - 1.6 cm and 2 parts by volume of gravel fraction 0.5 - 1 cm, the crushed PET and keramzite being mechanically mixed together, gravel is then added as the top and bottom layers or as the front and back layers of the filler, depending on the direction of groundwater flow, see Figure 2, which shows:

Λ position 1 - five to six parts by volume crushed PET size fraction 3 * 5 cm position 2 - two to three parts by volume of expanded clay - ceramite fraction 0.8 ^ 1.6 cm λ

position 31 - one volume fraction of gravel fraction 0.5 ^ 1.0 cm - upper / front part and / position 32 - one volume fraction of gravel fraction 0.5 * 1.0 cm - bottom / rear part.

Crushed PET bottles are a waste product that is inexpensive and, due to its surface properties, also suitable for the formation of a bio film layer by degraders originating mainly from the group of G bacteria. At the same time, the addition of crushed PET bottles reduces the porosity of the carrier composition compared to plain ceramite, which is advantageous in terms of increasing the total area available for bio film formation, while reducing the residence time of the groundwater in the reactor charge.

In the case of a vertical flow of decontaminated groundwater through the barrier - flow from bottom to top - it is necessary to insert the mixed charge into the reactive barrier in a cassette of inert material - stPP, and the groundwater flow must be led to one physically delimited place. , respectively. more cartridges with mixed charge are stored, see Figure 3. For horizontal flow, the mixed charge can be placed in a simple ditch, which dams the groundwater flow, in addition to inserting it into the reactor, see

I 4 * 6 *

Figure 1. In both cases, it is necessary, based on the hydrogeological assessment of the site, to locate the charge in a suitable location so as to capture as much of the contaminated groundwater as possible.

It is possible to use both natural - autochthonous and introduced allochthonous, bacterial population depending on biodegraded organic substance, resp. mixtures of substances. In order to achieve optimal growth of biofilm on the mixture and to ensure high degradation efficiency of organic contamination, it is advisable to apply in particular the following bacterial strains of the group G 'of bacteria: Achromobacter xylosoxidans DEK 2, Citrobacter freundii DEK 1C, Comamonas acidovorans ANI 1, Cupriavidus metallidurans DEK IR B1, Pseudomonasputida 161, Pseudomonas veronii 145, V1-2 and V2-1 and Rahnella sp. DEK 1, available from the bacterial collection of DEKONTA, a.s. The inoculation - recovery - of the mixed filler with these degraders must be carried out as follows:

1) to transfer the bacterial strain, respectively. strains from lyophilized form to 1 ml of sterile saline - sodium chloride 6 g / l, distilled water,

2) then transfer 100 μΐ of the thus prepared solution to petri dishes and then cultivate in a thermostat at 30 ° C for 48 hours,

3) subsequently transfer three loops of the cultured bacterial strain, respectively. strains to

150 ml of sterile nutrient medium - peptone 0,5 g / l, normal tap water - and continue to cultivate for 72 hours on a shaker at room temperature 21 ± 0,5 ° C,

4) transfer the cultures thus prepared quantitatively into a 5-liter glass bottle containing a non-sterile nutrient medium - peptone 0.5 g / l, common tap water - and then cultivate for 48 hours under continuous aeration with air oxygen at 21 ± 0 room temperature 1 ° C,

5) subsequently transfer quantitatively the entire contents of the storage bottle to 50 liters of starting medium contaminated groundwater taken from the rehabilitated site diluted with common tap water in a 1: 1 volume ratio - and further cultivate for 24 hours under continuous aeration with air oxygen at 21 ± 0.5 ° C,

6) then inoculate the mixed charge in the barrier, the inoculum being quantitatively poured directly onto the animated charge and cultured therein preferably in a closed circuit, ie without the groundwater flowing through the barrier for 48 hours under the temperature conditions of the rehabilitated site,

I 4

7) Finally, after 48 hours, take a sample of the cartridge for analysis of phospholipid fatty acid content, hereinafter referred to as PLFA, the inoculation being considered successful if the PLFA values of the G bacteria used are at least 150 ng / g mixed fill.

In order to accelerate the growth of the bio film, it is also advantageous to apply N and P in a weight ratio of 1: 0.6 to 1.2 in the form of a mineral fertilizer, in a concentration of 1 to 3.5 g / l.

In the case of low dissolved oxygen content, hereinafter DO, in purified groundwater values below 0.5 mg / l, it is advisable to provide additional aeration of the purified water by upstream aeration chamber 61 with the aeration segment located at the bottom in front of the actual biodegradation section according to Figure 4, showing:

position 1 - five to six parts by volume of crushed PET size fraction 3-5 cm position 2 - two to three parts by volume of expanded clay - ceramite of fraction 0,8-1,6 cm position 3 - two parts by volume of gravel fraction 0,5 ^ 1 , 0 cm - upper / front and lower / rear part position 4 - cartridge pack of inert material - stPP position 5 - barrier of inert material - stPP position 61 - aeration - aeration chamber position 62 - biodegradation chamber filled with mixed charge 1 , 2,3 position 7 - aeration grate located on the bottom of the reactor

The aeration chamber 61 will allow the DO to increase in the recovered groundwater to values of 6-7 mg / L, which are values that improve conditions for aerobic bacterial processes performed by obligatory aerobic and facultative anaerobic microorganisms specified in accordance with the present invention.

With respect to the G group of bacteria applied according to the method of the present invention, it is essential that the contamination at the remediation site be of organic origin in the absence of heavy metals. It is especially advantageous if they are aromatic and polyaromatic compounds, which may have one or more carbon atoms substituted by C1-, CFL, NH 2, NO ₂ - and OH, i.e. chlorobenzenes, chlorophenols, aniline, nitrobenzene, nitrotoluene , phenol, cresol, naphthalene, anthracene, phenanthrene.

Example 1: Evaluation of bio film formation on selected materials

In laboratory conditions, bio film formation was evaluated on five selected materials - two types of activated carbon, zeolite, crushed expanded clay and crushed PET - by t 4 4

I I 1> 8 total protein content on their surface. Lowry and Folin-Ciocalteus reagents were used to determine proteins in solution.

Initially, the initial protein content of each material, which can be determined in the medium after autoclaving, was determined. Of the test materials on its surface, no proteins contained one type of activated carbon, GAC S835, prior to inoculation. Protein traces not exceeding 0.11 mg / g carrier were found in other materials - GAC K835, zeolite, liadrain - crushed keramzite and crushed PET.

Protein content on the surfaces of the individual carriers was determined again after 10 days of cultivation of the test materials in 50 ml of basic mineral medium, hereinafter referred to as BSM from the term "basic salt medium, composition 0.17 g / 1 KH ₂ PO ₄ , 0.13 g / 1 K ₂ HPO ₄ , 0.71 g / l -NH 4) ₂ SO ₄ , 0.34 g / l MgCl ₂ · 6 H ₂ O and 1 ml trace element solution, with addition of 400 mg / l phenol and Cupriavidus degrader inoculum metallidurans DEK IR at normal laboratory temperature 21 ± 0,5 ° C. Protein content on the surface of tested carriers decreased in a number: liadrain - crushed expanded clay -> crushed PET -> zeolite -> both types of activated carbon - GAC K835 and S835. The results are presented graphically below, see Chart 1. These results correspond well to the course of the biodegradation assay, see Example 2.

In the case of liadrain, the production of bio film was also confirmed by image analysis - color photographic documentation is available. Unfortunately, crushed PET interacts with the dye used in image analysis, and therefore it is not possible to use imaging techniques to verify the production of bio film.

Example 2: Effect of carrier presence on phenol biodegradation

In the first series of laboratory tests, fed-batch cultivation of Cupriavidus metallidurans DEK IR was performed in the presence of liadrain - crushed keramzite, crushed PET and zeolite. The course of phenol degradation during cultivation is shown graphically below in Chart 2. The initial phenol concentration in the medium did not exceed 400 mg / l, phenol biodegradation proceeded very quickly in all variants. Phenol biodegradation in medium with individual carriers was compared with phenol biodegradation in the control variant, which contained no carrier, in this case only inoculated BSM medium. During the experiment, there was a gradual slight increase in phenol concentration at the beginning of each cycle, due to the addition of a stock of equal concentration to the culture flasks at different phenol biodegradation rates. Due to this gradual increase in phenol concentration, it was possible to detect differences between individual carriers at the end of the test. During the first two cycles occurred

I t1 ι »ι. . 1 τ ♦ »* 'r 1. 9 to phenol biodegradation approximately equally fast in all variants except for the control variant - without the carrier where the phenol utilization was slowest. Thus, the presence of carriers had a positive effect on the course of biodegradation. In the last cycle, when the initial phenol concentration was at the highest of about 600 mg / l - the control variant decreases the metabolic activity of the Cupriavidus metallidurans degrader, and the phenol degrades much more slowly. The presence of zeolite resulted in a slight increase in phenol degradation rate. The fastest phenol was utilized when liadrain and PET were present in the medium. At this point it should be remembered that none of these materials - liadrain, PET, zeolite - absorbs phenol, while strong sorbent is activated carbon. Comparison of the tested variants confirmed the decreasing rate of biodegradation in the series: liadrain - crushed expanded clay -> crushed PET -> zeolite -> control variant without carrier addition.

In parallel to the cultivation of Cupriavidus metallidurans in the presence of the above three materials, the variant with activated charcoal GAC K835, which has a high sorption ability to phenol, was also tested. Since the maximum sorption capacity of this material is very high experimentally determined by 268.6 mg of phenol per g of this material, there was a very rapid decrease in phenol concentration in the medium after dosing fresh medium. Only at the end of the experiment the initial phase of biofilm formation was observed on the carriers, but to a lesser extent than with other materials. The explanation for this behavior is that all the added amount of phenol was rapidly adsorbed onto the surface of the GAC K835 used, making the contaminant less readily available for bacterial metabolism, which was then unable to form the necessary biofilm layer on the carrier surface.

Example 3: Biodegradation of aniline in the presence of carrier-bound biofilm

Biodegradation of aniline was monitored under laboratory conditions in Erlenmeyer flasks containing 100 ml BSM media, selected carrier - as described in Example 2, in addition GAC S835 coal and aniline at an initial concentration of 250 mg / L were tested. The bio film of the bacterial degrader Comanumas acidovorans ANI 1 was pre-grown under these conditions for one and a half months. In parallel to the biodegradation test, adsorption experiments were carried out in Erlenmeyer flasks containing 50 ml BSM medium and 1 g of the respective carrier. The initial aniline concentration was the same as in the previous case, the final pollutant concentration was determined after 5 days.

The results of total losses, including the determination of the proportion of biodegradation and sorption, are shown in Table 1. The results show that carriers are the most suitable in terms of total aniline loss

- ιο GAC S835 and K835, where 95% loss of aniline was detected. However, much of the loss - 70% - was due to the adsorption of pollutant to this material. The highest aniline loss caused by degradation - 36.5% - was determined in crushed PET. The lowest rate of aniline degradation was achieved in the carrier - crushed liadrain, when the aniline loss was only 14.4%.

Also after completion of the experimental aniline degradation experiments, the total amount of proteins was determined, again according to the Lowry method, see Table 2. Protein concentration varied between carriers and the highest protein concentration was measured for crushed PET and liadrain - crushed keramzite.

Example 4: Biodegradation of polyaromatics including evaluation of the effect of mineral fertilizer addition

Glucose was used as a simple substrate that best characterizes the growth progression of a given taxon's microbial cells, since it is an easily utilizable source of carbon and energy. The growth rate on glucose was then compared with the growth rate on selected polycyclic aromatic hydrocarbons, hereinafter referred to as PAH - anthracene, phenanthrene and naphthalene. Evaluation of the growth rate of the test degrader Ochrobactrum anthropi NAF B1 was carried out by determining the optical density values (O.D.) at 400 nm over time.

The results confirmed that the bacterial population is able to utilize all three PAHs as primary substrates, see Chart 3. The increases achieved on individual PAHs are consistent with the structure and tabulated values of the solubilities of naphthalene, phenanthrene and anthracene, ie. the more water-soluble the pollutant is, the easier it is to transport and incorporate it into the metabolic pathways of the cellular organism and translates quantitatively into growth curves.

The study of the effect of the mineral content was monitored using the Bioscreen microcultivation device, where the ability to grow microorganisms in the medium of the given composition was monitored, see Table 3. Growth was expressed as the change in optical density - O.D. - measured automatically every hour in the white light spectrum 420 to 580 nm. Cultivations were carried out at 28 ° C for 7 days. Cultivation was performed in wells, pipetting of culture medium and inoculum was added to each well, at the same time 1 to 2 naphthalene crystals were placed in each well.

If the nitrogen source concentration was kept constant - 0.71 g / l - and the N: P concentration was chosen in the ratio 1: 0.2 - medium M2 - and lower - Ml Ochrobactrum anthropi NAF

B1 did not grow. In contrast, if the ratio N: P was 1: 0.4 - medium M3 - and higher -M4

Ochrobactrum anthropi NAF B1 grew well on naphthalene. If the concentration of the source P was kept constant - 0.3 g / l - and the concentration of the source N: P in the ratio 1: 1.7 - medium

M6 - and higher - M5, Ochrobactrum anthropi NAF B1, again due to unfavorable growth * 4 1 4 f ff · 4-1 «

- 11 conditions did not grow. By reducing the N: P ratio to 1: 0.8 - M7 medium - suitable cultivation conditions and good growth of Ochrobactrum anthropi NAF B1 were achieved, but at the same time cell deaths occurred virtually without a stationary growth phase.

Advantageous conditions for the cultivation of Ochrobactrum anthropi NAF B1 in terms of the presence of minerals were media with a ratio N: P -1: 0,0,9, with respect to the achieved concentration of bacterial biomass - OD _max about 0,38 - and in comparison with the amount of chemicals consumed .

Similar laboratory tests to those described in Examples 1-4 above were performed with all bacterial degraders of the present invention. The experimental results achieved were similar and led to the same conclusions.

Example 5: Effect of dissolved oxygen on biodegradation of real contamination

In the first phase of the laboratory experiments, the residence time of the liquid in the packed column system was tested at the same volume of injected oxygen - 60 l / h. Two different time intervals of 24 and 38 hours were selected and the ability of the two columns to remove the organic load at given time intervals was monitored. The tests were carried out on real ground water from the chemical industry contaminated mainly by chlorinated derivatives and nitroderivatives of aromatic substances, tested degraders were isolated from this waste water and it was Achromobacter xylosoxidans DEK 2 and Rahne Ha sp. DEK 1.

Based on the results of the first phase, it was found that with a shorter residence time of the liquid in the system, four to six types of substances could still be detected at the outlet, the largest share being recorded for nitrobenzene. In the case of a longer residence time, only two types of substances that could be considered to be difficult to degrade, namely nitro benzene and Ν, Ν-diethylaniiin, were detected at the output. The removal of these substances is subject to too abiotic effects and therefore these substances have been selected as suitable markers for the biodegradation activity of the systems.

In the second part of the laboratory tests, the amount of oxygen injected was reduced over time by reducing the inlet air volume from 50 l / h to 10 l / h over two weeks while maintaining a uniform residence time of 38 h. these experiments are recorded in Table 4.

Experiments with real water clearly showed that when trying to aerobic way of degradation of individual contaminants, it is advantageous to keep the dissolved oxygen concentration at the highest possible values - about 7 mg / l. In our case it was. 12 this concentration is ensured at air flows of 40 l / h and above, which approximately corresponds to the values observed during fermenter cultures. When the air flow dropped to 30 l / h it was possible to notice negative changes in most of the monitored parameters, ie pH at the outlet of the columns increases, respiratory activity of microorganisms decreases, the numbers of microorganisms bound to the carrier show a decrease and last but not least and N, N-diethylaniline.

Example 6: Quarterly operation simulation of the filling barrier operation

The aim of the column tests was to verify biofilm formation and biodegradation efficiency in a continuous system on a quarter-scale scale under conditions that simulate groundwater remediation conditions at the site. The chemical site contaminated by mixed organic pollution - especially chlorinated benzenes, monohydric phenols and chlorophenols, aniline, nitrobenzene, nitro toluene, naphthalene - was chosen as a model site.

The column system consisted of three glass double-shell columns. Water flowed between the two shells, which cooled the entire system to a temperature of 11 ° C ± 0.5 ° C simulating the average temperature of the aquifer at the selected model location. Each column had a separate supply of contaminated water, which was provided by a membrane pump. The arrangement of the contaminated water and aeration gas - air flow was countercurrent, the contaminated water inflow was situated at the top of the column, aeration gas - air - was fed to the bottom of the column as shown in Figure 5, indicating:

positions 1, 2, 3 - medium tested

position 4 position 5 position 6 position 7 position 8 - cooling water supply - cooling water outlet - aeration gas supply - a container of contaminated water - recovery tank

Three types of materials were used for packing the columns: activated carbon of fraction 0.5-1 cm, liapor-uncrushed ceramic clay of fraction 0.8-1.6 cm and PET cut into size fraction 1-2 cm. Due to the physical properties of keramzite and PET, which are difficult to sink below the water surface, it was necessary to add a total of 20% by volume of gravel - fraction 0.5-1 cm. The gravel was divided into two equal portions and then placed at the top and bottom of the columns. Between these two portions of gravel was the actual test material, ie granular activated carbon - column 1 - and expanded clay mixed with PET in a 1: 1 volume ratio of columns 2 and 3.

Columns 1 and 3 were inoculated with a mixture of four selected Comamonas acidovorans ANI 1, Cupriavidus metallidurans DEK IR, Ochrobactrum anthropi NAF B1 and Pseudomonas veronii 145, Column 2 was inoculated with natural microflora originating from the contaminated site, Achromobans xKosoxidox xoxosox degraders. DEK 1. The inoculation of the columns was carried out as described in the present invention. The content of phospholipid fatty acids - PLFA - of the used G 'bacteria on the carriers after 48 hours of cultivation was 235 ng / g AU, 261 ng / g liapor - keramzite and 412 ng / g PET.

By comparing the efficiency of biodegradation of the contaminants - RE,% - present in the waste water and the biodegradation rate - q, mg / l.h - depending on the increasing organic load - OL, mg / l.h, the optimum flow of contaminated water through the bed was determined. Determination of optimal flow rate, resp. The hydraulic retention time - HRT, h - then made it possible to design the necessary residence time in the model system of the pilot plant barrier, which was subsequently implemented directly in the real conditions of the Pardubice locality - Example 7.

Column 1, i.e. a column filled with activated carbon, inoculated with 4 selected alochtonic degraders, showed the highest resistance to the total organic load pumped on the column during the test. Chart 4 on the left shows a relatively high degradation efficiency - RE - columns over the entire test period and a gradual reduction of the total organic load - OL, which was given by changing parameters of the groundwater withdrawn, on the right the load characteristics of this column. The results show that with decreasing amount of organic substances at the input - decreasing OL - there was a gradual decrease of degradation efficiency and linear decrease of degradation rate - q It follows that decrease of concentration of contaminants in waste water leads to decrease of column efficiency due to the lack of substrate used by the microorganisms present to grow.

Since this column was packed with high sorption capacity, higher than expected efficiency was expected. According to the calculations, the packing contained in the column should not be fully saturated during the test and the contamination elimination efficiency should thus be almost 100% during the test. On the other hand, after about two months of the test, it was found that the purified water at the exit of the column was no longer absolutely clear as it was at the beginning, but began to stain similarly to the contaminated water at the inlet of the column. In this case, biodegradation predominated over passive sorption of contaminants and activated carbon then served as a biomass carrier, as confirmed by the results of PLFA analyzes. The content of G 'degraders on the AU surface increased after 4 months of experiment to values of 6 569 ng / g, an increase of about 28x and is approximately the same as measured in columns 2 and 3 for expanded clay.

Compared to column 1, column 2 did not achieve such high efficiencies from the start, due to the different type of packing - a mixture of ceramics and PET - and the fact that only biodegradation contributes to the reduction of contamination and that the adsorption rate is zero. The degradation efficiency showed an uneven course, partly corresponding to the course of the total organic load. It is also possible to read lower efficiency values * 6 to 56% from the load characteristics of this column - Chart 5. In contrast, the observed dependence of degradation efficiency and degradation rate was similar to that of column 1, ie with decreasing total organic burden, degradation efficiency and degradation rate gradually decreased, but in this case non-linear. Again, it is not possible to expect a high efficiency of this column at the current flow rate and very low concentration of contaminants at the inlet. Groundwater with organic substances at a concentration of approximately 15 mg / l.h should be supplied to achieve more than 50% degradation efficiency. The reason for this is the fact that the autochthonous bacteria present have been phenotypically adapted to these high levels of organic pollution.

Given the 100% biodegradation rate, there was also a higher increase in the PLFA G content of bacterial groups after 4 columns of the column experiment. In the case of liapor, it was up to 6,972 ng / g - an increase of 27x-a PET up to 15,147 ng / g - an increase of 37x. PLFA analysis also confirmed that G 'bacteria were the predominant group of microorganisms on carriers, and that microflora grew much better on PET than keramzite and activated carbon. The results of the genetic analysis of ribosomal DNA further confirmed that the biofilm production in Column 2 was mainly due to the autochthonous bacteria Rahnella sp. DEK 1 from Achromobacter xylosoxidans DEK 2.

Column 3 inoculated with allochthonous strains showed similar behavior to that of the column

2. DNA analysis confirmed that out of the 4 added allochthonous strains only two survived on the carriers, ie Comamonas acidovorans ANI 1 and Cupriavidus melallidurans DEK 1R. However, after two months, these two strains have also disappeared, replaced by the natural population of Rahnella sp. DEK 1 AAchromobacter xylosoxidans DEK 2.

Furthermore, degradation efficiency of contaminants in individual columns was compared. The monohydric phenols were eliminated by column 1 with an efficiency of 92-98%, with adsorption to the surface of activated carbon almost exclusively. The course of phenol removal in the other two columns was similar, after the initial lower degradation efficiency - 23-25% - at the end of the test the 60-70% concentration of this substance was removed. Benzene, toluene, ethylbenzene and xylenes - BTEX were almost 100% degraded in column 1. Adsorption again played a major role here, but due to the high .15 ´ volatility, also 10 to 30% of the content of these substances flowed into the air - the flow rate was measured by the sorption tubes. For the other two columns, BTEX removal rates and volatilization rates were similar, ie 72 ^ 83% biodegradation and 17-28% effluent. The same behavior was shown for chlorobenzenes and dichlorobenzenes, where due to lower volatility there was no significant outflow together with exhaust air, the ratio of biodegradation to outflow in columns 2 and 3 ranged from 84 to 99% to 1 to 16%. Aniline and nitrobenzene were biologically degraded in columns 2 and 3 of 89 * 96% and 40 * 85%. In the case of column 1, these substances were also removed, but again there was no biodegradation, but adsorption of contamination.

Example 7: Pilot Verification of a Packing Barrier

The biological barrier with the subject matter of the present invention has been practically tested at a model locality in Pardubice. On the basis of hydrogeological survey of the locality, a technical design of the barrier and reactor layout was developed. Due to the high level of groundwater fluctuation at the site, a drainage system was chosen for verification, which allowed better capture of contaminated groundwater into the barrier reactor. After purification, the groundwater was then seeped into the rock environment by a soaking gallery, thereby achieving a minimum level of backwater behind the barrier reactor.

A chamber reactor system divided into three chambers according to Figure 6 and located below the terrain was used as the reaction segment of the barrier at the model site. Due to the low dissolved oxygen content in the groundwater, two aeration chambers were placed in front of the two parallel filling chambers, see Figure 4. The filling chambers were filled with a mixture of coarse crushed PET - 5 volumes - and expanded clay - 3 volumes - mixed together and then supplemented with upper and lower proportion of gravel - 2 volumes. From the location of the gravel fractions, it is evident that the flow rate through the filling chambers was from bottom to top according to Figure 3, in which it indicates:

position 1 - five to six parts by volume of crushed PET size fraction 3-5 cm position 2 - two to three parts by volume of expanded clay - ceramite of fraction 0,8-1,6 cm position 3 - two parts by volume of gravel fraction 0,5-1 , 0 cm - divided into upper and lower part position 4 - cassette cover of inert material - stPP position 5 - barrier reactor made of inert material - stPP

For the other parts of the barrier, the flow was horizontal. Piezometers placed on the bioreactor made it possible to monitor the groundwater level in the collecting drain, individual chambers and also to carry out sampling of water at the inlet and outlet of individual chambers.

♦ * * ♦ ♦ t * * * * * * * * * * *

- 16 Inoculation of the filler was not performed, autochthonous bacterial population - especially Achromobacter xylosoxidans DEK 2 and Rahnella sp. DEK 1 - addition of mineral NP fertilizers in concentration 1 g / l. Dissolved concentration

The G ^- oxygen was maintained in the range of 7-9 mg / L.

The results of the post-operation test confirmed the conclusions of laboratory and quarter-stage tests. By its behavior the filling chamber III approached the most conditions. Barriers, see Chart 6. Organic chamber load ranged from 0.07 to 30.72 mg / l.h, with the "recommended" OL concentration above 15 mg / l.h being maintained during the first 70 days of the -V test. The level of contamination corresponded to the degradation efficiency - 24-34% - even with the rate * in biodegradation - 4.48- ^ 10.28 mg / l.h. The good efficiency of the chamber in the first phases of the pilot test is clearly linked to the increase in cultivable microorganisms and hence the available autochthonous pollution degraders, see Chart 7. The decrease in efficiency occurs after this period due to a decrease in organic load below 15 mg / l.h.

PLFA analyzes showed different contents of total biomass in the filling chambers. In the case of chamber III. the biofilm growth was lower, but its nature - the predominance of G * bacteria and the orderly higher biofilm growth measured on PET - compared to liadrain - was the same in both packed chambers. The presence of the native strain of Achromobacter xylosoxidans DEK 2 was also confirmed by ribosomal DNA analysis in both packing chambers, see Figure 7.

At the end of the pilot experiment the measured data of pollution reduction in individual chambers of the biological barrier were compared, contamination decay constants k, h ¹ were determined - according to the 1st order decomposition model, then the half-lives of contamination decay were calculated - t | _{/ 2} , h. Experimentally determined half-lives showed unambiguously positive effect of filling addition on pollution degradation, in chambers II. and III. the disintegration time of most sanitized substances was significantly shortened. The only exception are two nitro compounds, which are hardly degradable substances. Here, the addition of the carrier had neither a positive effect nor a negative effect on its degradation rate.

The above-mentioned practical applications - Examples 1 to 7 - mixtures of crushed PET, expanded clay and gravel and selected G degraders, combined with the addition of minerals and oxygen to the sanitized groundwater, have proven their utility within the biological fill barrier technology as effective for acceleration of biodegradation of selected organic pollutants through the formation of stable biofilms.

Table 1: Total loss of aniline, including specification of loss due to biofilm of Comamonas acidovorcms ANI 1 and adsorption to the surface of the carrier used

Material, carrier 4- Total loss |%] Adsorption 1%] Biodegradation [%] liadrain - crushed expanded clay 17.0 2.6 14.4 zeolite 35.0 5.3 29.7 crushed PET 44.7 8.2 36.5 GAC S835 95.0 69.0 26.0 GAC K835 95.8 70.1 25.7

Table 2: Total amount of proteins on carriers

Material, carrier Proteins [mg / g | crushed PET 0.397 liadrain - crushed expanded clay 0.252 zeolite 0.139 GAC S835 0.130 GAC K835 0.134

Table 3: Composition of media used to study the effect of N and P content on the biodegradation of naphthalene by Ochrobactrum anthropi NAF B1

Source P and N - »Medium - N: P 4 P-power supply K ₂ HPO ₄ + KH ₂ PO ₄ [g / l] N - source (NH ₄ ) ₂ SO ₄ [g / l] 1 - 0 0.00 0.71 M 2 -1: 0.2 0.15 0.71 M 3 -1: 0.4 0.30 0.71 M 4 - 1: 0.9 0.60 0.71 M5 -0: 1 0.30 0.00 M6 - 1: 1.7 0.30 0.18 M 7 - 1: 0.8 0.30 0.36 M8 - 1: 0.2 0.30 1.42

-ι8Table 4: Effect of aeration rate on biodegradation of organic matter in real waste water

Aeration rate Parameter outputs both columns 4 50 l / h 401 / h 301 / hr 20 l / h 10 1 / h nitrobenzene [resid. conc. %] 2 1,2-2 4 3,6-4 9-11 Ν, Ν-diethylaniline [resid. conc. % 3-15 5-8 18 20 May 6-6,5 DO concentration [mg / 1] 6.7-6.9 6.7-6.8 6-6,2 5-5,3 3,7-3,8 CO ₂ production [mg / 24h.l] 502-555 581.4 329-423 423-444 338-423 number of micro-organisms water [KTJ / ml] 10 ⁶ -10 ⁷ 10 ⁴ -10 ⁵ 10 ⁴ -10 ⁵ 10 ⁵ 10 ” number of microorganisms fill [KTJ / g] 10 ⁶ 10 ⁷ 10-10 ’ 10 ⁵ -10 “ 10--10 ⁵ pH 4-4,5 5.4-5.7 6.5-6.6 4,4-5,4 3,3-4,3

Table 5: Half-lives of contamination in individual barrier chambers

Biobarrier Chamber -> Contaminant Φ I. aerace 11. refill 111. cartridge BTEX 990 231 231 chlorobenzene 1 155 347 231 dichlorobenzene 1 155 347 347 nitrobenzene 347 347 347 aniline 693 693 693 naphthalene 693 231 231 monohydric phenols 231 173 173 nonpolar extractables 1 386 173 173

- Industrial applicability of the invention

The mixed fill and the method of its inoculation with selected bacterial strains mentioned in the invention is widely used industrially in the field of remediation of old ecological burdens, especially in the field of in situ remediation of contaminated groundwater. Compared to other biological permeable reactive barriers, the process specified in the invention utilizes a novel mixed biofilm carrier medium which, by reviving a precisely defined group of G 'alochtonic degraders, resp. Combined with an autochthonous microflora, it is able to positively support the biodegradation of contaminated groundwater flow while reducing operating costs and reducing remediation time. The inventive method is particularly suitable for remediation of aromatic and polyaromatic compounds present in the contaminated ground water with the pollutants which may have one or more carbon atoms substituted by C1-, CH ^ -, NHz-, NO ₂ - and OH These are primarily chlorobenzenes, chlorophenols, aniline, nitrobenzene, nitrotoluene, phenol, cresol, naphthalene, anthracene, phenanthrene.

Claims (4)

Patent claims A carrier material for biofilm formation in packed biological barriers for the remediation of groundwater contaminated with organic compounds, characterized in that it comprises a blend of crushed plastic waste bottles made of polyethylene terephthalate * ι PET> ♦, supplemented with a certain proportion of expanded clay * ceramiczihria gravel, it contains 5 to 6 parts by volume of crushed PET size fraction of 3 ^to 5 cm, 3 to 2 parts by volume of 0.8 to 7% fraction of the ω-i-expanded ceramite, and Support material according to claim 1, characterized in that, for the vertical flow of purified groundwater through the barrier, the support material is embedded in a stabilized polypropylene cassette package (stPP). 2 parts by volume of a gravel fraction of 0.5 * 1 cm, the crushed PET and expanded clay being mechanically mixed together, and the gravel forms the upper / front and lower / rear fill layers depending on the direction of groundwater flow. A carrier material according to claim 1, characterized in that it comprises at least one degrader inoculum selected from Gram negative + G; * bacterial groups including Achromobacter xylosoxidans DEK 2, Citrobacter freundii DEK 1C, Comamonas acidovorans ANI 1, Cupriavidus metaliidurans DEK IR, Ochrobactrum anthropi NAF B1, Pseudomonas putida 161, Pseudomonas veronii 145, VI-2 and sp2-1 and V2-1. DEK 1. 4. The support material of claim 1, further comprising nitrogen 4N; * and phosphorus * P / ⁱⁿ a weight ratio of 1: 0.6 to 1.2 in the form of a mineral fertilizer, in a concentration of 1 to 3.5 g / l.