FI85984C - Microbiological purification of water and therefore used microorganism - Google Patents

Description

1 85984

Microbiological purification of water and the micro-organism used for it

Microbiological preparation of water from the same microorganism

The invention relates to a method for purifying contaminated water, in particular raw or surface water and more particularly also groundwater, from microbiologically degradable contaminants. The invention also relates to a novel microorganism for use in the method as such and immobilized on a solid support.

In industrialized countries, chemically contaminated water is becoming an increasingly serious environmental problem. Pollution released into the environment as a result of industrial activity, waste and damage can be very toxic even at very low concentrations, and in many cases nature is unable to decompose these problematic pollutants or decomposes them only very slowly. Such problematic pollutants include, for example, chlorinated phenolic substances, polycyclic aromatic hydrocarbons, oil, various solvents, biocides, etc. The quality of raw water is also impaired by other types of impurities that are difficult to remove and can be considered as pollutants in this context. Pollution is e.g. sought to eliminate them by biological decomposition, taking advantage of the k.o. microorganisms specializing in decomposition.

In particular, chlorinated phenolic substances and their derivatives, for which the term "chlorophenols" is used for brevity in the following description and claims, constitute a low-degradable and highly toxic problem pollutant in nature. Many fish species die as long as the concentration of pentachlorophenol (PCP) in the water is 0.6 mg / l or less. Most of the commercially produced chlorophenols are used to impregnate 2,859,884 wood, and soils and water bodies contaminated with chlorophenols have been widely found, especially around wood impregnation plants. Chlorophenols are also used in many biocides and, for example, in photographic chemicals. Chlorophenol contamination has been observed in both surface waters and groundwater at concentrations that are toxic to the environment.

In the biological treatment of wastewater, chlorophenols pose a problem not only due to their polluting effect but also by killing other microorganisms involved in the treatment. The biodegradation of chlorophenols in wastewater treatment has been reported by Etzel et al., Dev. Ind. Microbiol. (1974) 16: 287-295, in which wastewater containing 20 mg / l of pentachlorophenol (PCP) was slowly passed in a continuous stream through the biomass of a microorganism growing on the fiber wall of the reactor. Most of the pentachlorophenol had decomposed after a residence time of 6 hours. A wastewater treatment system that degrades chlorinated phenols has also been described by Portier et al., Toxicity Assessment: An International Quaterly (1986) Vol. 1, pp. 501-513. In the system, PCP metabolizing microorganisms were immobilized on a solid support that was either chitin, glass wool, or cellulose. The carrier did not substantially adsorb chlorophenols. The effluent containing 100 mg / l PCP was passed as a continuous stream through the reactor and the PCP decomposed satisfactorily to a level of about 1 mg / l in 6 to 7 hours.

Purification of contaminated soil from chlorophenols (approx.

400-500 mg / kg) by composting using chlorophenols to decompose the induced microorganism Rhodococcus chlorophenoli-cus PCP-1 DSM 43826 have been reported by Valo et al., Appl. Microbiol. Biotechnol. (1986) 25:68 ... 75.

However, the second problem with contaminated surface and groundwater is the huge amount of material treated and the relatively low concentration of toxic substance compared to contaminated wastewater or soil, where the amount of 3 85984 substances treated is limited and the amount of contamination is generally relatively high.

According to U.S. Pat. No. 4,713,340, chlorophenol-contaminated surface and groundwater have been treated using chlorophenol-degrading bacterium ATCC 39723, which is capable of degrading chlorophenols. to an acceptable level. Although, according to the publication, the bacterium is capable of removing up to 100 mg / l of PCP from lake water in 40 to 75 hours, it can be easily understood that the purification of entire lakes and contaminated groundwater in breeding ponds is not industrially viable. Brown et al. Appl. Environ. Microbiol. (1986) Vol. 52 pp. 92-97, have proposed the use of said bacterium, Flavo-bacterium sp., In combination with natural bacterial strains attached to natural stone for the purification of river waters when the PCP content is up to 600 mg / l. Also in this case, the treatment of surface and groundwater is a problem due to the amount of water to be treated.

In addition, in many cases the temperature of the surface and especially the groundwater is so low that it does not correspond to the preferred operating temperature of the microorganisms (usually about 20 ... 35 ° C). Heating large amounts of water to the operating temperature of microorganisms is an energy consuming activity, in addition to which heated water can cause additional problems in nature.

It is an object of the invention to provide a method by which large amounts of water can be efficiently purified from biodegradable contaminants.

It is also an object of the invention to provide a biofilter comprising an immobilized microorganism useful in the method.

4,85984

It is a further object of the invention to provide a particularly advantageous, isolated, purified microorganism for use in the method.

More specific objects of the invention will become apparent from the following description and appended claims.

Accordingly, the invention relates to a method for the microbiological purification of contaminated water by means of a decomposing microorganism, characterized in that the water to be purified is passed through a collecting biofilter so that the water is substantially purified and enriched in the filter, and the microorganisms are immobilized on the biofilter. to decompose the accumulated pollution.

The invention is particularly preferably applied to the removal of chlorophenols from contaminated water, in which case the invention also relates to a solid support for use as a biofilter, which comprises microorganisms which degrade chlorophenols. The carrier is preferably a large surface area porous organic material which reversibly collects chlorophenols from water and in which chlorophenol degrading microorganisms are immobilized.

In a preferred embodiment of the invention, chlorophenol-degrading bacteria belonging to the genus Rhodococcus are used as immobilized chlorophenols which are capable of removing chlorophenols released into the water from the biofilter. As a novel microorganism, Rhodococcus sp. CP-2, which has been isolated from nature for the first time and is now presented in its purified form.

The invention is illustrated by the following description and the accompanying •. with the aid of figures in which 5 85984

Figure 1 shows an applied reactor in which the process according to the invention can be applied.

Figure 2 shows another reactor alternative for applying the process according to the invention.

Figure 3 shows a graph of the degradation of pentachlorophenol and the formation of carbon dioxide, respectively, due to the chlorophenol degrading activity of the microorganism according to the invention.

Figures 4 show curves of the total amount of chlorophenol fed to the reactors and the chloride formed in the application of the process according to the invention.

Figure 5 shows the biodegradation of chlorophenols when applying the process according to the invention.

Figure 6 shows the amount of chlorophenol leaving the reactor in the chlorophenol contaminated water purification experiment.

In the method according to the invention, the contaminant is purified from water by enriching it in a biofilter and thus very large amounts of water, as well as surface or groundwater, can be purified per unit time, even from relatively low but still harmful amounts of contaminants. The residence time of the water flowing through the biofilter can be very low, as the time required for relatively slow biodegradation does not have to be taken into account during filtration. Thus, the cleaning efficiency of the equipment per unit time is many times higher than the amount of water that could be cleaned in the same space and at the same time by direct biodegradation of the pollutant. The equipment may be small, even if the amount of water to be treated is large.

- * The ability of bacteria to decompose pollutants, and especially the rate of decomposition, is usually temperature dependent. The problem in surface and 6 85984 groundwater treatment is often that at the feed-water temperature, about 4 ... 20 ° C, the bacterial activity is not at its maximum. Thus, if the feedwater temperature and / or other conditions reduce the degradative activity of the bacteria, biodegradation is said to be absent during the filtration step.

Within the scope of the invention, it has been found that a method which is particularly suitable for purifying large amounts of water from slowly decomposing pollution can be obtained by a two-step method. In the first stage, the biodegradable contaminant is continuously removed from a large amount of flowing water by enriching the contaminant in a biofilter. In the second step, the microorganisms immobilized on the biofilter are caused to decompose the contaminant accumulated in the biofilter in a relatively small amount of water.

In the second, biodegradation step of the process, a small amount of water surrounding the filter can be recycled, allowing the water to be easily heated to a preferred level of biodegradation, generally to about 20-35 ° C. It is clear that if the method is applied to the treatment of warm water or the bacterial activity does not require a warm environment, no separate heating is required.

Thus, if the feed water temperature is favorable and other conditions are suitable, the immobilized bacteria can act as a decomposer during the filtration step and decompose the contaminant as it accumulates in the biofilter. In this case, the decontaminated water and decomposition products from bacterial biodegradation. The substances formed as a result of the biofunction may be either harmless, water-soluble substances, or they may adhere to the biofilter in the manner of the original contaminant, in which case such a substance formed in the biofunction is preferably decomposed by the decomposing microorganism. If harmful decomposition products dissolve in water due to biodegradation, care must be taken to ensure that no decomposition occurs during filtration or that decomposition products must be removed by other means.

The supply of water to be purified through the biofilter is stopped at the latest when the biofilter is saturated with contamination, which can be observed from the increase in the concentration of contaminants in the flowing water, i.e. from the fact that the biofilter starts to leak contamination. In practice, however, it is usually not advisable to saturate the biofilter with contaminants. The contaminant concentration of the filter at the time of interruption may depend on the microbial tolerance, especially in the case of toxic contamination, in which case the water supply must be interrupted before the contamination concentration in the biofilter exceeds both the immobilized microorganism tolerance and the biofilter saturation point.

Once an appropriate amount of contaminants has accumulated in the filter, the water supply is interrupted and a relatively small amount of water can be recycled in the biofilter. At the same time, care is taken to ensure that the environmental conditions in the biofilter are as favorable as possible for the bacterial activity that decomposes the pollution. Thus, the temperature, pH, possible nutrients, etc. are set at a level favorable to this activity and constant efforts are made to keep each bacterium in the range required for bioactivity.

Suitable a pollution-collecting biofilter according to the invention is any pollutant-collecting substance which can be removed; at the same time, it is able to act effectively as a substrate for the adhesion of degrading microorganisms. Such substances which collect chemical compounds generally have a high specific surface area and a high porosity. For example, porous organic substances generally work well as biofilters according to the invention. Examples of biofilters for chlorophenols are polyurethane resins and wood chips and wood bark, the polyurethane being found in the process according to the invention -. the lowest priced. The polyurethane resin may be in a floating form in water or it may be a special water-heavier polyurethane modified to immobilize bacteria. Modified poly-6 85984 urethane pulp is the preferred biofilter because it binds chlorophenols due to its large surface area and surface charge. At the same time, the pulp is an effective substrate for microbial attachment.

Bark grits and wood chips have been found (see Apajalahti et al. Microbiol. Ecol. (1984) 10: 359 ... 367) to absorb chlorophenols and are also suitable biofilters for chlorophenols, as they reversibly bind chlorophenols from solutions containing them and also work well as an attachment medium for organisms.

Microorganisms useful in the method are microorganisms that decompose the pollutant to be removed and are capable of immobilizing on a carrier that acts as a biofilter. Microorganisms that selectively degrade each problem contaminant are known in the art and one skilled in the art will be able to select from these. where appropriate, the most appropriate micro-organism.

Examples include chlorophenol-degrading microorganisms, such as some Arthrobacter and Pseudomonas bacteria, as described in Portier et al., Toxicity Assessment: An International Quarterly, Vol. 1, p.

501-513, (1986), and as pure cultures of Flavobacterium sp.

(ATCC 39723), described in U.S. Patent 4,713,340, and Rhodococcus chlorophenolicus PCP-1 (DSM 43826), described in Apajalahti et al. Int. J. Syst. Bacteriol., (1986) 36: 246 ... 251.

In addition, within the scope of the invention, a hitherto unknown microorganism, Rhodococcus sp. CP-2, deposited with the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) on 13 May 1988 in accordance with the Budapest Treaty and numbered DSM 4598.

9 85984

Microorganisms Rhodococcus sp. CP-2 was isolated from chlorophenol-contaminated soil. A slurry was formed from a soil sample into a mineral salt solution to give a PCP-degrading mixed culture. The culture was enriched by adding PCP (5 mg / l) several times, after which one PCP-degrading colony was selected from the culture growing on an agar plate containing PCP (20 mg / l) and given the designation CP-2.

Rhodococcus sp. The CP-2 strain has the characteristics of the genus Nocardioform actino-mycetes and is considered to belong to the genus Rhodococcus. CP-2 grew on the plate as an orange-yellow, mucous colony and had a cook-rod-cook cell morphology during the growth phase. Visible colonies (1 ... 4 mm in diameter) appeared after one week of culture. Growth occurred at 18-37 ° C, no growth at> 45 ° C.

The optimum growth temperature was 28 ° C. Growth occurred in 0.003 and 3.0% NaCl, but not 7% NaCl. Growth of 1% glucose, fructose, mannitol, maltose (weak), sorbitol, trehalose (weak), inositol and mannose as carbon source. Methanolysis of the cells released the following simple fatty acids (relative proportion in parentheses): C10: 0 (3), C14: 0 (6), C16: 0 (23), C16: lcis9 (2), C16: ltrans9 (9), C18: 0 (1), C18: ltrans9. . (19) and IOCH3Cl8 (tuberculostearic acid) (12). The cell wall contained mycolic acids with a length of 32 to 36 carbon atoms. Menaquinones contained 9 isoprenoid units with a single hydrogenated double bond (type MK-9H2).

CP-2 is able to use chlorinated phenols as its sole carbon source and removes 10 μM PCP in 5 hours to a concentration of 1 μg / l. Strain CP-2 also degrades several other tetra-, tri-, and dichlorinated phenols, guaiacols, and syringols and does not lose its ability to degrade chlorophenols when transferred to a chlorophenol-free medium. Rhodococcus sp.

; CP-2 as well as the previously isolated Rhodococcus chlorophenolicus PCP-1 (DSM 43826) are particularly suitable bacteria for water purification by the process of the invention, as they are able to purify water almost completely from chlorophenols released by biofilter (see e.g. Figure 3). . It will be appreciated that chlorophenol degrading strains developed from these bacteria as well as other microbial strains having similar degradation capacity may also be used in the method.

The aerobic degradation of pentachlorophenol to carbon dioxide and inorganic chloride by bacteria belonging to the genus Rhodococcus is shown by the following reaction scheme: s & '- «-Sr - & m—

Cl OH w OH QH OH

In this context, it should be noted that chlorohydroquinones are formed as the first decomposition product of chlorophenols. These substances are used, for example, as photographic chemicals and can thus be removed from the wastewater of the photographic industry by the method according to the invention.

Bacteria useful for the decomposition of contaminants other than chlorophenols are known in the art and can be used in suitable embodiments of the process of the invention. It is also possible to similarly isolate other contaminating bacteria from nature, for example contaminated soil or water, as well as other chlorophenol-degrading bacteria which can be used in the applications of the method according to the invention.

In the method according to the invention, the decomposing microorganisms are used in immobilized form attached to a solid support. The degrading bacteria are preferably allowed to grow in the nutrient solution together with the solid carrier-forming mass, whereby the bacterium is attached to the carrier. Attachment can be based on, for example, adsorption or covalent bonding. In the case of immobilization of 11,859,884 strains of Rhodococcus which decompose chlorophenols on a support such as polyurethane resin or bark, the microorganisms attach to the support by means of mucus formed when they grow using sugar as a carbon source.

The ability of Rhodococcus CP-2 and Rhodococcus chlorophenolicus PCP-1 to decompose chlorophenol is genetically stable but requires induction. The ability of bacteria to degrade contaminants can be induced either before immobilization or after immobilization. In connection with the induction, the bacteria are fed a chemical to be decomposed in connection with the nutrient solution so that the bacteria produce the pollution-degrading enzymes.

In practice, the biomass can be immobilized on a support by circulating the pure culture stock grown in the fermentor through the support mass until a sufficient amount of biomass is attached to the support.

A carrier comprising an immobilized microorganism belonging to the genus Rhodococcus which degrades chlorophenols is a new product developed according to the invention. Preferred carriers for the applications according to the invention are organic porous substances, such as bark, wood chips and modified polyurethane resins. Microorganisms of the genus Rhodococcus immobilized on a carrier retain their ability to function for a long time. This is a great advantage in terms of their commercial use.

Since the biodegradation of chlorophenols results in the formation of hydrochloric acid, the reactor circulating fluid must be buffered during the decomposition phase. Instead, there is no need to add nutrients to the reactor, as the microorganisms in the reactor are able to use chlorophenol-derived carbon as their sole carbon source.

i2 85984

If necessary, the reactor biomass can be activated or regenerated after each or more biodegradation steps by circulating nutrient-containing water in the reactor. The reactor is preferably fed to the reactor. sugar sources that favor the growth of the microorganism. Preferably, the bacteria are fed a carbon source which favors the desired bacterial strain as well as possible, but which as few other bacteria as possible are able to use. In this case, the desired microbes increase and a little detached biomass is observed in the water leaving the reactor. In contrast, during the decomposition and filtration phase, the biomass is not substantially released from the support.

After or during resuscitation, it may be necessary or at least advantageous to re-induce the degradation mechanism of the contaminant in the microorganism. This is then done by introducing a small amount of contaminants, such as chlorophenol, into the reactor to initiate the biosynthesis of the degrading enzyme.

The reactor can be cleaned if necessary, for example by pressure washing with a recirculation pump or, in the event of a possible blockage, by inspecting those suitably in the reactor! through the filling hatches.

The process is preferably applied in a reactor system with at least two reactors, one operating in the filtration step of the process at the same time as the other undergoing the biodegradation process. The method can be advantageously applied in a system in which several reactors are connected in parallel or in series, so that any reactor can undergo a biodegradation process while the other reactors act as biofilters.

If there are more than two reactors in the system, the treatment steps of the reactors are recycled periodically so that the reactor which has accumulated the longest contamination is subjected to the decomposition step and the reactor that has undergone the decomposition step in the previous section.

Figure 1 illustrates a reactor suitable for the purification of chlorine-phenolic water. This bioreactor comprises several overlapping compartments 1 filled with a mass acting as a microbial attachment surface. The microbes on the surface of the pulp are capable of decomposing chlorophenols. The pulp is filled into the reactor through the filling hatches 2. The pulp is placed on perforated midsoles 4, which support the filling mass and divide the reactor space into different compartments. Air is introduced into the reactor via the aeration connection 5 if the degrading action of the microbe is an aerobic process and requires aeration. The water to be purified is led via a line 10 by means of a feed and recirculation pump 7 through a feed and recirculation pipe 8 to a sprinkler 6, which dispenses the water to be cleaned evenly into the reactor.

The upper end of the reactor has a biodegradation gas and aeration gas outlet pipe 12 and the lower end of the reactor has a purified water outlet pipe 11. Each compartment has sampling taps 9 and in addition the reactor has a number 3 monitoring level indicating the liquid level. The reactor recycle tube 8 further includes non-shown thermal resistors that can heat the circulating water to the desired level required for microbial biodegradation. The pipe 8 may further have a valve-closed supply line for any buffer and / or nutrient solution to be fed, which nutrient solution is fed to recyclable water, especially during the reactor recovery stage.

There may be several such reactors in series or in parallel in the reaction system so that the water supplied by the pump (or pumps) can be led to and from any reactor to another or out of the system by suitably turning the valves. In this system, any or any of the reactors may be excluded from the feed water line, 14 85984, whereby water can be circulated through the circulation pipe, respectively.

The process according to the invention can also be applied in the reactor shown in Fig. 2, in which the support 1 comprises microbial-degrading microbes attached to its surface. The feed water is led to the reactor via an inlet line 4 at the bottom thereof by means of a feed and recirculation pump 3. After passing through the biofilter 1, the purified water enters the clarification space 2 at the upper end of the reactor and the water is further discharged through the outlet pipe 5.

For the aerobic operation of the microbe, the reactor is aerated through a perforated aeration line 6 in the lower part of the reactor and the reactor gases exit through a pipe 7.

However, the following examples further illustrate the invention without, however, limiting it in any way.

Example 1

Isolation of the decomposing microorganism 10 500 g samples of contaminated soil or sludge were inoculated into 500 ml columns containing softwood bark as a solid support and 200 ml of mineral salt solution was percolated through the column. Tetrachloroglycol (TeCG) was added weekly to a concentration of 10-50 μM. After three months, it was found that the percolation fluids contained mixed cultures that repeatedly removed added TeCG (10 μM). Cultures obtained from percolators were enriched with several dilutions and fed with 10 to 20 TeCG at intervals of 2 to 3 days. Culture samples were plated on DSM-65 (glucose 4.0 g, yeast extract 4.0 g, malt extract 4.0 g, H 2 O 1000 mL, pH 7.2) agar containing 10 μM TeCG, and 130 colonies from each mixed culture were tested for TeCG degradation by measuring the disappearance of TeCG from solution. TeCG degrading cultures were purified.

«85984

By a similar method using pentachlorophenol as the enrichment substrate, e.g. Rhodococcus CP-2 (DSM 4598), which completely removed 10 μΜ pentachlorophenol (PCP) in five hours and 70% of 1λ0: 11θ labeled PCP was excreted as 14CO2 (see Figure 3). Figure 3 shows the degradation of 10 μΜ PCP containing C-PCP as a function of time, with the symbol · showing the PCP concentration and the symbol O showing the formation of evolved 14CO 2.

Strain CP-2 also degraded several other chlorinated phenols, guaiacols, and syringols in 10 μΜ solutions. In these experiments, pentachlorophenol (PCP), 2,3,4,5-tetrachlorophenol (2345-TeCP), 2,3,4,4,6-tetrachlorophenol (2346-TeCP), 2,3,5,5,6-tetrachlorophenol (2356 -TeCP), 2,3,4-trichlorophenol (234-TCP), 2,3,5-trichlorophenol (235-TCP), 2,3,6-trichlorophenol (236-TCP), 2,4,5- trichlorophenol (245-TCP), 2,5-dichlorophenol (25-DCP), tetrachloroguaiacol (i.e. tetrachloro-2-methoxyphenol) (TeCG), 3,4,5-trichloro guaiacol (345-TCG), 3, 4,6-trichloroguaiacol (346-TCG), 3,5,6-trichloro guaiacol (356-TCG), 4,5,6-trichloroguaiacol (456-TCG), 3,4-dichloro gua a coli (34-DCG), 3,5-dichloroglycol (35-DCG), 3,6-dichloroguaiacol (36-DCG), trichlorosyringol (i.e. trichloro-2,6-dimethoxyphenol) (TCS) and 3,5-Dichlorosyringol (35-DCS) decomposed in 48 hours.

Other pollutant-degrading bacteria or other chlorophenol-degrading bacteria which can be used in the process according to the invention can also be isolated by a similar method.

A: Example 2

Immobilization of the microorganism on a carrier

Pure cultures of chlorophenol-degrading micro-organisms, •. Rhodococcus chlorophenolicus PCP-1 (DSM 43826) and Rhodococcus »» * * CP-2 (DSM 4598) were immobilized on a polyurethane resin mass, i.e. 85984, a polyurethane resin REA 90/16 manufactured by Bayer Ag, West Germany, which is modified to suit bacterial immobilization (particle size less than 10 mm; density at 20 ° C 1.14 ... 1.05 kg / l; weight in suspension 115 kg dry matter / m3 suspension volume; sedimentation rate: 94 + ^ 6 m / h).

The mixed culture of bacteria was grown in a fermentor in the presence of 1% glucose, 1% sorbitol as a carbon source in the presence of a carrier. As they grew, the bacteria secreted mucus and noticeably adhered to the surface of the carrier material. When biomass formation was sufficient, cultivation was stopped.

The polyurethane mass comprising the immobilized Rhodococcus culture was stored for 20 days at + 4 ° C. In an experiment performed after storage, it was found that the microbes are still able to degrade chlorophenols.

Chlorophenol-degrading Rhodococcus bacteria have been found to degrade chlorophenols even after one year of storage at + 4 ° C.

Example 3 Bioreactor operation

The experiments were performed in a 0.5 L laboratory scale experimental reactor with a water supply port and a degassing port at the top, a water outlet at the bottom, and an aeration port at the bottom. Modified polyurethane REA 90/16 (manufactured by Bayer Ag, West Germany) was used as the carrier. The amount of pulp in the reactor was 400 ml.

Rhodococcus chlorophenolicus (DSM 43Θ26) and Rhodococcus CP-2 (DSM 4598) were used as scattering microbes. The microbes were immobilized on a polyurethane resin support by growing them in a suitable growth solution with the resin.

i7 85984

The experimental reactor was filled with active resin immobilized on both microbial species. The identical control reactor (sterile) was without microbes. A sterile parallel experiment was used to elucidate the binding of chlorophenols to the surface of the pulp and to eliminate the possible role of non-biological mechanisms in degradation.

Chlorophenol-containing water with a chlorophenol content of either 0.1, 5, 10 or 20 mg / l was used as the feed water. The water supply rate was 2 vol / d. The temperature was + 25 ° C, aeration rate 5 ml / min. Mild buffering was performed with phosphate buffer to maintain the pH at about 7.

In connection with the experiment, the decomposition of chlorophenols was observed: 1) as an increase in the amount of inorganic chloride in the water passed through the biofilter. The chloride was derived from the bound chlorine of the chlorophenol molecule; 2) by adding 14 C-labeled pentachlorophenol to the reactor, it was shown that C 1 D carbon dioxide from the labeled PCP ring escaped with the exhaust gas; 3) by measuring the total chlorophenol content of the effluent and the feed water by gas chromatography; 4) comparing the analytical results of the active biofilters and the sterile biofilter, only the active column was found to degrade chlorophenols.

Chlorophenols (in the form of the wood impregnating agent Ky-5, manufactured by Kymi Oy, Kuusankoski) were passed through the reactors according to Table 1 below for 63 days. The chlorophenol content was measured from the feed solution by gas chromatography.

ie 85984 TABLE 1.

Concentration of chlorophenol (Ky-5) in the solution fed to the biofilters during the experiment

Time (days) Chlorophenol content (mg / l) 0.. . 1 1 3,4 1 1 ... 20 6, 2 20.. . 29 12, 8 29.. . 40 20, 3 40.. . 50 0 50. . 53 25, 3 53.. . 58 0 68.. . 62 25, 3 62.. . 0

The average chlorine phenol composition of the Ky-5 pesticide is shown as a percentage by weight in Table 2 below.

TABLE 2.

Chlorophenol content,% 2, 6-DCP <0,01 2, 4-DCP 1 2, 4, 6-TCP 11 2, 4, 5-TCP 0,06 2, 3, 4-TCP 0,04 2, 3 , 4, 6-TeCP 80 PCP 7, 5

During the experimental period, the liquid fed had a very low nutrient content (N, P, etc.) so that microbes would not divide and the reactor would leak microbes.

At day seven, 14 C-labeled pentachlorophenol with randomly labeled ring carbon atoms was fed to the reactors to demonstrate mineralization of pentachlorophenol using ίΛ0-labeled carbon dioxide.

«« * * · «· I9 85984

Throughout the experimental period, the sterilized biofilter remained free of microbial growth. It did not emit radioactive carbon dioxide or inorganic chloride.

The test results are shown in Figures 4 ... 6.

Figure 4 shows the decomposition of chlorophenols in the biofilter to inorganic chloride. In the upper curve, the sum of the chlorophenol (CP) fed to the reactors during the experiment and the chloride (Cl-) formed in the lower curve. The chloride contained in chlorophenol is about 60% by weight. Based on this, the immobilized cells have lysed about 55% of the added chlorophenol in 63 days. The remainder of the chlorophenol is bound to the polyurethane support.

Figure 5 shows the biodegradation of pentachlorophenol by immobilized bacteria. At day 7 of the experiment, the added radioactive pentachlorophenol (ring carbons randomly labeled with * 4C) (55,500 cpm) decomposed into labeled carbon dioxide. Its yield was almost 50%. Some of the labeled PCP remained bound to the polyurethane mass within reach of the bacteria and a small portion may have been converted to cell mass. No radioactivity was found to enter through the decomposition reactor.

Figure 6 shows the amount of chlorophenol precipitated in each reactor. Both sterile and bacterial filters effectively bind chlorophenols for 25 days. Thereafter, the non-decomposing reactor begins to leak chlorophenols rapidly. The degradable biofilter, on the other hand, permeated only a small amount of chlorophenol, with the concentration level in the effluent water generally remaining below 10 μg / l, which is the permissible limit for the chlorophenol content in domestic water. The concentration in the effluent of the sterile filter increased to 1 mg / l at the end of the 60-day test period due to the saturation of the polyurethane mass with chlorophenols.

20 85984

Example 4

Experiment with cold feed water

The same equipment as in Example 5 was used in the experiment. Cold (+ 5 ° C) chlorophenol-containing water was fed through the reactor for three days and a slight decrease in chlorophenol binding capacity was observed.

The reactor was then brought to room temperature (+ 25 ° C) at which point its decomposition capacity immediately started. The amount of chloride in the recycled liquid increased strongly as an indication of the decomposition of chlorophenol.

During the continuous experiment (experiment continued for 98 days), cold water purification was tested several times in the reactor described above. In this case, the reactor was fed with the amounts of chlorophenols in water shown in Table 3 for 2 or 1 day, after which the water supply was stopped and the water was circulated at +25 ° C in the reactor for 5 and 16 days, respectively. The experiment parameters and test results are shown in Table 3.

During the cold treatment, the amounts of chloride in the liquid that passed through the test and reference reactors were equal, but when the heat was introduced into the test reactor, the amount of chloride in the liquid was higher than in the reference reactor, ie microbial activity starts as soon as the temperature is favorable.

The invention has been described above mainly as a suitable method for removing chlorophenols. However, it is clear to a person skilled in the art that a similar method can also be applied to other biodegradable contaminants, which can be attached to the degrading microorganisms in question. to a biofilter that collects pollution.

21 85984 3

C/O

4-1 rH O LD I — I

: 0J (N CM rH CO

CD --4 · —I

<#> i (0

‘ΓΛ <8 U (Ö W

s 5%

3 --H jC O CTt CO

4J Ό --4 Π »VO rH in D --4 <0 e aM> O 10 (Ό -H 3> Ji 41 • r4 l-l

O

44

Hi (0 (0 in rH 4 »m to I 0) ro co o o

tn ID l-1 CO o rH OH

·· o 3 CO 4T CO rH

y h h

0 3 -rH

-h nj m m -η 4-1

OH WO 1H

+ 8 S 81 ID $ lu r 3 5t <£ 5 8 I Ϊ

«5 O H * O

X --4 j * 4J

4J J * rf 4 »00 H

C ft <3 (0 oo ov cm m to U> φ

φ Q dl U

ä J 3 8: <3 <4-1 jo • 1 “

C 1 I

s it ~ «; 8 g - (-: ro i-H 4— to Hi: <3 tn -η σι rH o 4j * W: rtj> 1 rH 41 · 00 o 00 I I 3 8 * 01 I £ 3 3 £

V

O U

--4 O>

4-> --H

0 4.1 10

Ci ft to 3

8 O -HO

e r sv c f5 x § <01: m r— 4J: m vo a ifiSJ <<^ <

CO o>,> «> ·, CM CM r-H CM

o 8 «- £ ^

SUH

--4 Ji o 2 O Q) a \ m o r *

rH O 0% O H H

X ££ «H» H »H

22 85984

Degradability of chlorophenols of bacteria CP-2 and PCP-1

Each bacterial strain degrades several polychlorinated phenols. In both, the ability to degrade is inducible, i.e., degrading enzymes are formed in the bacterial cell only when a chlorinated phenol is present in the culture and protein synthesis is possible at the same time.

Decomposable chlorophenols. The following table shows the degradability of chlorophenols of both bacteria in liquid culture with a chlorophenol concentration of 10 μM. Degradation expressed as a percentage of the initial concentration.

Chlorophenol PCP-1 CP-2 PCP 100 100 2345- TeCP 20 100 2346- TeCP 100 100 2346-TeCP 100 100 234- TCP 60 100 235- TCP 100 100 236- TCP 100 100 245- TCP 20 100 246- TCP 30 80 345-TCP 10 90 23- DCP 40 80 24- DCP 10 50 25- DCP 40 100 2 6-DCP 30 40 34- DCP 10 80 35- DCP 30 80 23 85984

Other characteristics: 1) growth with different carbon sources (+ = increases, - = does not increase)

Carbon source PCP-1 CP-2

Glc weak + fructose + + mannitol + + maltose - weak sorbitol + + trehalose + weak inositol + + mannose - +

The following fatty acids are released by bacterial methanolysis, in brackets their relative proportions:

Fatty acid PCP-1 CP-2 C 10: 0 - 3 C 14: 0 24 6 C 15: 0 12 C 16: 0 100 23 C 16: 1 20 11 C 18: 0 12 1 C 18: 1 88 19 IOCH3C28 67 12 C 20: 0 4 C 22: 0 4 C 24: 0 5

Mycolic acids C33-C43 C32-C36

In the dish, PCP-1 and CP-2 form slimy orange-yellow colonies. The colonies of both strains are slightly different.

PCP-1 forms visible colonies on yeast extract agar and rhamnose agar for 1-2 weeks at 28 ° C. CP-2 forms visible colonies within 1 week.

Claims (17)

A process for microbiological purification of contaminated water by means of a microorganism or microorganisms which degrades chlorinated phenolic substances and / or derivatives thereof, i. chlorophenols, in which process the water to be purified is passed through a biofilter and microorganisms immobilized in the biofilter break down the chlorinated compounds, characterized in that the purification is carried out essentially as a two-phase process, using a solid porous as a biofilter carriers which amplify enriched chlorophenols from the aqueous medium and serve as a basis for the chlorophenol-degrading microorganisms, wherein in the first or filtration phase the water to be purified is measured through the biofilter to enrich chlorophenols therein, and in the second or biodegradation phase the flow of feed water through the process. is disrupted and the chlorophenol-degrading microorganisms immobilized in the biofilter are caused to degrade enriched chlorinated compounds. Process according to claim 1, characterized in that the relatively small amount of water surrounding the biofilter is circulated in the biofilter during the biodegradation phase. 3. A method according to claim 1 or 2, characterized in that the parameters of the water surrounding the biofilter are adjusted during the biodegradation phase to obtain or maintain a favorable environment for the degrading microorganisms. Method according to claim 3, characterized in that the parameter being adjusted is the temperature of the water. 28 85984 Process according to any of the preceding claims 1, 4, characterized in that the water soin is to be purified, water soin is colder than the activity temperature of the degrading microorganisms, such as surface or groundwater, the temperature of the filtration phase being kept at the temperature of the supplied water. , and that at the beginning of the biodegradation phase, the temperature of the water surrounding the biofilter is raised to a level favorable to the activity of the microorganisms, usually about 20. . 35 ° C and maintained at this temperature until the enriched chlorinated phenolic substances and / or derivatives thereof have substantially decomposed. Method according to any of the preceding claims 1, 5, characterized in that bacteria belonging to the genus Rhodococcus are used as degrading microorganisms. Method according to claim 6, characterized in that as bacteria bacteria Rhodococcus sp. CP-2 (DSM 4598) and / or Rhodococcus chlorophenolicus PCP-1 (DSM 43826). Process according to any of the preceding claims 1, 7, characterized in that as a biofilter a porous substance is used that reversibly enriches chlorophenols, in which substance immobilized microorganisms which degrade chlorophenols, which substance is advantageously polyurethane resin, wood bark or wood chips. Method according to claim 8, characterized in that a biofilter is used for immobilization of microbes modified polyurethane resin, in which immobilized Rhodococcus bacterial culture (s) which degrades chlorophenols. Process according to any of the preceding claims 1 ... 9, characterized in that the biofilter is transferred, after a completed biodegradation phase, to the filtration phase by entering water to be purified, and that the chain of operation consisting of the first and second phases 28 85984 is repeated. A method according to any of the preceding claims 1.. . 10, characterized in that there are two or more biofilters in series or in parallel, so that when a biodegradation is complete, the second or the other chlorinated phenolic substances filter out of the supplied water. Method according to claim 11, characterized in that water is supplied through two or more in series connected biofilters and that the biofilter that was first in the flow order is transferred in the biodegradation phase and the biofilter that has been in the biodegradation phase is last brought into the flow order for supply of water. Process according to any of the preceding claims 1 ... 12, characterized in that the microorganisms immobilized in the biofilter are regenerated at certain intervals after an effected biodegradation phase by circulating nutritious water through the biofilter, so that the microorganisms multiply. A pure culture of a microorganism which degrades chlorinated phenolic substances and / or derivatives thereof, i. chlorophenols, in aqueous medium, which pure culture promotes are immobilized in a solid porous carrier which enriches chlorophenols, characterized in that the microorganism is Rhodococcus sp. CP-2, DSM 4598, which is induced to degrade chlorophenols. A solid porous support useful in the process of claim 1 and which enhances enriched chlorinated phenolic substances and / or derivatives thereof, i.e. chlorophenols, from an aqueous medium and act as a support for microorganisms which degrade chlorophenols, characterized in that the carrier is a large surface polyurethane resin which enriches chlorophenols reversibly and in which immobilized a mixture or pure culture or cultures of microorganisms which degrade chlorophenols. 30 85984 Carrier according to claim 15, characterized in that the carrier is a micro-organism modified polyurethane resin modified for immobilization and that the micro-organism is a culture or cultures of microorganisms of the genus Rhodococcus. Carrier according to claim 16, characterized in that, as microorganisms, Rhodococcus chlorophenolicus PCP-1 (DSM 43826) and / or Rhodococcus sp. CP-2 (DSM 4598)