A PROCESS FOR THE MICROBIOLOGICAL PURIFICATION OF WATER AND A MICROORGANISM FOR USE IN SAID PROCESS

Abstract: The invention relates to a process for the microbiological purification of water which is polluted by contaminants such as chlorophenols. The purification is performed with the aid of microorganisms which degrade the contaminant in question, whereby the feed water is directed through a biofilter which captures the contaminant so that the water is essentially cleaned and the contaminants are enriched in said filter. Microorganisms which have been previously immobilized on the biofilter are made to degrade the contaminant captured in said biofilter. The purification of huge amounts of water is quicker and the size of the apparatus smaller when the relatively slow biodegradation can be performed as a separate second step of process. The process is especially suitable for the purification of cold waters, which normally are difficult to purify. In the present process only the water circulating in the biofilter during the biodegradation stage needs to be heated to a temperature which is suitable for bioactivity. The invention also relates to a novel microorganism which can be used in said process as well as to a solid support containing immobilized chlorophenol-degrading microorganisms which can be used as a biofilter.

Description: A process for the microbiological purification of water and a microorganism for use in said process The invention relates to a process for the purification of polluted water, especially raw or surface water and more specifically also ground water from contaminants which are microbiologically degradable. The invention also relates to a novel microorganism which can be used in said process as well as to said microorganism immobilized on a solid support. Chemically polluted water is becoming an increasingly serious environmental problem in the industrialized countries . Contaminants which have ended up in the environment in con¬ sequence of industrial activity, wastes and damages can be very poisonous even at very low concentrations and in many cases such hazardous contaminants are not naturally degraded at all or degrade only very slowly. Hazardous contaminants are for example chlorinated phenolic compounds, polycyclic aromatic hydrocarbons, oil, various solvents, biocides, etc. The quality of raw water is also impaired by other contami¬ nants which are not easily removed and which in this con¬ nection can be regarded as contaminants . Efforts have been made to remove the contaminants e.g. by biodegradation utilizing microorganisms with a specific capacity of degrading specific contaminants. Particularly chlorinated phenolic compounds and their derivatives, which are referred to by the term "chlorophenolε" for short in the following specification and claims are extremely toxic and hazardous contaminants, which are hardly degradable at all in the environment. Several types of fish will be killed even when the concentration of pentachloro- phenol (PCP) in the water is 0.6 mg/1 or less. Most of the commercially produced chlorophenols are used for wood preservation and the soil and waterways particularly around wood preservation sites have been found to be polluted by chlorophenols. Chlorophenolε are also used in many biocides as well as in for instance photographic chemicals. Chlorophenolic pollution has been found both in surface waterways and ground waters at concentrations which are toxic for the environment. In the biological purification of wastewater the chlorophenolε pose a problem not only because of their polluting effect but also by killing the other purifying microorganisms. Etzel et al. in Dev. Ind. Microbiol. (1974) 16:287...295 have reported on the biodegradation of chlorophenolε in the puri¬ fication of waεtewater, whereby wastewater containing 20. mg/1 of pentachlorophenol (PCP) was passed in a slow continuous flow through the biomasε of a microorganism growing in the fiber,-wall of a reactor. Most of the pentachlorophenols had degraded after a detention time of 6 hours. A chlorophenol- degrading system for the purification of wastewater has also been disclosed by Portier et al. in Toxicity Asseεεment: An International Quarterly (1986) Vol 1, p. 501...513. In said system the PCP metabolizing microorganisms were immobilized on a solid support of either chitin, glass-wool or cellulose. The support did not adsorb chlorophenols to any appreciable extent. Waεtewater containing 100 mg/1 of PCP was passed in a slow continuous flow through the reactor, and the PCP was satisfactorily degraded to a level of about 1 mg/1 in 6 to 7 hours . Valo et al. in Appl. Microbiol. Biotechnol. (1986) 25:68...75 have reported the purification of polluted soil from chloro¬ phenols (about 400...500 mg/kg) by composting utilizing the microorganism Rhodococcus chlorophenolicus PCP-1 DSM 43826 which had been induced to degrade chlorophenolε. In connection with polluted surface and ground waters the vast amount of material to be treated and the relatively low concentration of the toxic material, however, poses another problem compared to polluted wastewater or soil, wherein the amount of the treated material iε limited and the amount of contaminants in general is relatively high. In US Patent 4.713.340 an effort has been made to purify εurface water and ground water polluted by chlorophenols utilizing bacteria ATCC 39723 of the genus Flavobacteriu , which are capable of degrading chlorophenols. Bacteria induced to degrade PCP were added to basinε containing the polluted water, and the bacteria were cultured in εaid water until the chlorophenol concentration had decreaεed to an acceptable level. While the patent teacheε that the bacteria are capable of removing up to 100 mg/1 of PCP from lake water in 40...75 hourε 1 , it will eaεily be realized that the purification of whole lakeε and polluted ground water in culture basins is not industrially feasible. Brown et al. Appl. Environ. Microbiol. (1986) Vol. 52 p. 92...97 have εuggeεted the uεe of the said bacteria, Flavobacterium sp. together with natural bacterial εtrainε fixed on rock for the purification of river waters when the concentration of PCP was as high aε 600 mg/1. Alεo in this case the purification of εurface and ground waters poses a problem owing to the quantity of water to be treated. Moreover, in many caεeε the temperature of εurface water and especially ground water is so low that it does not correspond to the favourable temperature of activity (generally about 20...35 °C) of the microorganisms. Heating large quantities of water to the temperature of activity for the microorganisms consu'mes energy, besideε which the heated water may have additional hazardous effects on the environment. The object of the present invention is to provide a process, by which large quantities of water can be efficiently purified from biodegradable contaminants. The object of the invention is also to provide a biofilter containing an immobilized microorganism, which filter is suitable for use in said process. The object of the invention is further to provide a novel microorganism, which is isolated from the environment and purified and which preferably can be used in εaid process. The more detailed objects of the invention will appear from the following specification as well as from the appended claims. Thus, the object of the invention is a proceεε for the micro¬ biological purification of polluted water by a contaminant- degrading microorganiεm, εaid process being characterized in that the water to be purified is conducted through a bio¬ filter which captures the contaminant so that the water is esεentially cleaned of the contaminant and the contaminant is enriched in εaid filter, and in that the microorganisms which are immobilized in εaid filter are brought to degrade the captured contaminant. The invention is preferably applied to the removal of chloro¬ phenols from polluted water, a further object of the invention in a preferred embodiment thereof comprising a solid support useful as said biofilter, which support contains chlorophenol- degrading microorganisms. The support preferably compriseε a porous organic material having a large surface area and being capable of reverεibly capturing chlorophenolε from water, said chlorophenol-degrading microorganisms being immobilized said support. In the preferred embodiment of the invention the microorga¬ nisms which degrade chlorophenolε and which are immobilized on the biofilter comprise chlorophenol-degrading bacteria of the genus Rhodococcus which are capable of very efficiently removing chlorophenols freed from the biofilter into the sur¬ rounding water. In the εcope of the invention the microorga¬ nism Rhodocoecus εp. CP-2 is disclosed as a novel microorga¬ nism which had not previously been isolated from its natural environment and which is herewith preεented aε a new isolate. The invention is further illuεtrated by the following speci¬ fication and the appended drawings, wherein Fig 1 repreεentε one embodiment of a reactor εuitable for uεe in the proceεs of the invention. Fig 2 represents an alternative reactor for the carrying out of the process of the invention. Fig 3 is a graphical representation of the degradation of pentachlorophenol and the evolution of carbon dioxide, respectively, in response to the chlorophenol-degrading action of the microorganism of the invention. Fig 4 are graphical repreεentationε of the total amount of chlorophenol fed into the reactors and the chloride produced in the practice of the proceεε of the invention. Fig 5 εhowε an indication of the biodegradation of chloro¬ phenolε in the practice of the proceεs of the invention. Fig 6 shows the amount of chlorophenol discharged from a reactor in a test for purifying chlorophenol-polluted water. In the proceεs of the invention the contaminant is removed from the water by enriching the contaminant into a biofilter and in this way it iε possible to clean very large amounts of water in a short space of time. It iε thereby possible to clean also surface water and ground water even from contami¬ nants at relatively low but still harmful levels. The retention time for the water flowing through the biofilter may be very short since in the filtration it is not necessary to provide the time required by the relatively slow bio¬ degradation. Thus the cleaning effect of the apparatus in a given time unit is many times higher than treating the cor¬ responding amount of water in the same space and the same time by a direct biological degradation of the contaminant. The apparatus may be small even though the amount of treated water is large. The activity and especially the degrading rate of the bacteria capable of degrading the contaminants generally depend on the temperature. One common problem in connection with the purification of εurface water and ground water relateε to the activity of the bacteria not being at itε maximum at the temperature of the feed water, about 4 to about 20 °C. Thus, in case the feed water temperature and/or other circumstances are such that they lower the degrading activity of the bacteria, hardly any biodegradation will take place during the filtration stage. In connection with the invention it was observed that a process which is especially suitable for purifying large amounts of water from recalcitrant contaminants can be provided by a two-stage method. In the first stage the bio¬ degradable contaminant iε continuoεly removed from a large amount of water flowing through the apparatuε by enriching the contaminant into a biofilter. In a second stage microorganisms immobilized on the biofilter are brought to degrade the con¬ taminant captured into the biofilter, in a relatively small amount of water. In the said second or biodegradation stage a small amount of water surrounding the filter may be circulated, whereby the water may eaεily be heated to a level which iε favourable for the biodegradation, generally to about 20 to about 35 °C. It is clear that in case the process is used for the purification of warm water or the bacterial activity does not require a warm environment, there is no need for any separate heating. In case the temperature of the feed water is favourable and the other circumstances are suitable, the immobilized bacteria may have a contaminant-degrading function already during the filtering stage and will degrade the contaminant as it is captured into the biofilter. Water which iε cleaned from the contaminant in queεtion will then be continuouεly diεcharged from the filter together with the contaminant metaboliteε. The metaboliteε formed as a result of the biological activity may either be harmless, water soluble compounds or they may become fixed on the biofilter in the same way as the original contaminant. In the latter case the contaminant-degrading microorganiεm will preferably degrade alεo εuch compounds formed by the biological activity. In case hazardous degradation products are disεolved in the water aε a reεult of the biodegradation it is necessary to ascertain that no degradation takes place during the filtration or the degradation products must be removed in some other way. The feeding of the water to be purified through the biofilter iε diεcontinued not later than when the biofilter is saturated with 'the contaminant, which saturation may be detected by the rise in contaminant concentration in the water flowing through the apparatus, i.e. by the fact that the biofilter begins to leak contaminant. In practice it is generally not, however, worth the while to saturate the biofilter with contaminant. Especially in the case of a toxic contaminant the contaminant concentration in the biofilter at the time of disconnection may depend on the tolerance level of the microbes, and the feeding of water should be discontinued well before the con¬ taminant level in the biofilter has risen above the tolerance level of the immobilized microorganiεm as well as the saturation point of the biofilter. When a suitable amount of contaminant has been captured in the filter the feeding of water is discontinued and a relatively small amount of water may be circulated in the biofilter. At the same time care should be taken to ensure that the ambient conditions in the biofilter are as favourable aε possible for the activity of the contaminant-degrading bacteria. Thus, the temperature, pH, any nutrients, etc. are adjusted to a level which is favourable for said activity and efforts are made to retain εaid level within the range required by the bioaσtivity of the bacteria in queεtion. In accordance with the invention the contaminant-capturing biofilter may comprise any material which captures the con¬ taminant to be removed and which is at the same time capable of acting effectively as an attachment εubεtrate for the degrading microorganiεmε. Such materials which capture chemical compounds generally have a large specific surface area and great porosity. For example porous organic compounds generally function well as biofilters in accordance with the invention. As biofilters for chlorophenolε, materials εuch as polyurethane resins as well as wood chips and wood bark may be mentioned, polyurethane being considered most preferable in the process of the invention. The polyurethane resin may be in a form which floats on the water or it may comprise poly¬ urethane which iε heavier than water and iε εpecifically modified for the immobilization of bacteria. The modified polyurethane mass is the preferred biofilter since it binds chlorophenols into itself because of itε large εurface area and itε εurface charge. Said maεε iε simultaneously an effective attachment subεtrate for microbes. Crushed wood bark and wood chips have been found to abεorb chlorophenols (see Apajalahti et al. Microbiol. Ecol. (1984) 10:359 - 367) and εaid materials are alεo suitable as bio¬ filters for chlorophenolε since they bind chlorophenols reversibly from solutions containing chlorophenols. Said materials function well as attachment εubεtrateε for micro- organiεms . The microorganisms which are useful in the present procesε are microorganiεmε which degrade the contaminant which is to be removed and which microorganiεmε are capable of being im¬ mobilized onto the biofiltering support. Microorganisms which selectively degrade any specific hazardous contaminant are known in the art and a perεon skilled in the art can choose from among these the most suitable microorganiεm for any given caεe. Exampleε of εuσh microorganiεmε include the chlorophenol- degrading microorganiεmε εuch as some Arthrobacter and Pseudomonas bacteria which have been disclosed by Portier et al. in Toxicity Aεεessment: An International Quarterly, Vol 1, p. 501 - 513, (1986), and aε pure cultures Flavobacterium εp. (ATCC 39723), which is diεcloεed in US Patent 4,713,340, as well as Rhodococcus chlorophenolicuε PCP-1 (DSM 43826) which iε diεcloεed by Apajalahti et al. in Int. J. Syεt. Bacteriol., (1986) 36:246 - 251. In connection with the present invention we have further isolated and purified a heretofore unknown microorganism, Rhodococcus sp. CP-2 which is deposited in accordance with the Budapest Treaty on May 13, 1988 into the depository DSM I (Deutsche Sammlung von Mikroorganiεmen und Zellkulturen GmbH) and received the number DSM 4598. The microorganiεm Rhodococcus chlorophenolicus PCP-1 (DSM 43826) was originally deposited in the DSM and has been continuosly available from said depository during the completion of the present invention. Said microorganism has also been re-deposited in accordance with the Budapest Treaty on January 10, 1989 in DSM and has been designated DSM 5128. Both above mentioned Rhodococcus bacteria are capable of degrading chlorinated phenolic compoundε and their derivatives but they differ εlightly in the amountε of degraded chloro¬ phenol. The novel microorganiεm Rhodococcuε CP-2 haε a some¬ what better capacity for degrading variouε other chlorophenolε than pentachlorophenol. The microorganiεm Rhodoσoccuε εp. CP-2 waε iεolated from chlorophenol contaminated εoil. The εoil waε formed into a slurry in a mineral εaltε εolution, wherefrom a PCP-degrading mixed culture waε obtained. The culture waε enriched by repeated additionε of PCP (5 mg/1). Then one PCP-degrading colony waε chosen from a culture growing on an agar plate containing PCP (20 mg/1) and waε designated CP-2. Rhodococcus sp. CP-2 has characteristics of the nocardioform actinomyσetes and it waε aεεigned to the genus Rhodococcus. CP-2 grew aε orange-yellow mucoid colonieε on the plate and in the incubation phaεe it had a coccoid-rod-coccoid cell morphology. Viεible colonieε (diameter 1 - 4 mm) appeared after one week of incubation. Growth occured at temperatures of 18 - 37 °C, no growth at >45 °C. The optimum growth temperature was 28 °C. Growth occured in 0.003 and 3.0 % NaCl, but not in 7 % NaCl. Growth on 1 % glucose, fructose, mannitol, maltose (weak), sorbitol, trehalose (weak), inosi- tol, and mannose aε the carbon source. The methanolysis of the cells released the following simple fatty acids (the relative abundance iε indicated in parenthesis) : C10:0 (3), C14:0 (6), C16:0 (23), C16:lcis9 (2), C16:ltrans9 (9), C18:0 (1), C18:ltrans9 (19) and IOCH3CI8 (tuberculostearic acid) (12). The cell wall contained mycolic acids having a length of 32 - 26 carbon atoms. The menaquinoneε contained 9 iεoprenoid units and one hydrogenated double bond (type MK-9H2). CP-2 is capable of using chlorinated phenolε aε its sole carbon source and it removes 10 uM PCP in five hours to a concentration of 1 g/1. The strain CP-2 alεo degrades several other chlorinated phenols such aε tetra-, tri- and dichlori- nated phenols, guaiacols and syringols and it doeε not loεe its capacity of degrading chlorophenols when being transferred to a substrate lacking chlorophenolε. The εtrain Rhodococcuε sp. CP-2 as well aε the previouεly iεolated Rhodococcuε chlorophenolicuε PCP-1 (DSM 43826) are especially suitable bacteria for the purification of water in accordance with the process of the invention since they are capable of cleaning the water almost completely from the chlorophenols being re- leased from the biofilter (εee e.g. Fig. 3). It iε evident that alεo chlorophenol-degrading strains derived from εaid bacteria as well aε other microbial strains having the cor¬ responding degrading capacity can be used in the present procesε. The aerobic degradation of pentachlorophenol to carbon dioxide and inorganic chloride by the action of bacteria of the genuε Rhodococcus is shown in the following reaction scheme: cl OH co 2 It should be noted that the first degradation products of the chlorophenols are chlorohydroquinones. Such compoundε are used for example as photographic chemicalε and they may thuε be removed from the waεtewaterε of the photographic induεtry in accordance with the proceεε of the invention. Bacteria which are useful for degrading other contaminants than chlorophenols are known in the art and they can be used in the implementation of the process according to the invention in suitable conditions. In a corresponding way it is also possible to isolate from the environment εuch aε from contaminated εoil or water bacteria which degrade other con¬ taminants as well as other chlorophenol-degrading bacteria, which can be used in the implementationε of the proceεs of the invention. In the proceεs according to the invention the contaminant- degrading microorganiεms are used in an immobilized form attached to a solid support. The degrading bacteria are preferably allowed to incubate in a nutrient medium together with a maεs compriεing the εolid εupport material, whereby the bacteria become attached to the εupport. The attachment may be based e.g. on adsorption or covalent binding. When it is a question of immobilizing chlorophenol-degrading Rhodococcus strains onto a support such aε a polyurethane reεin or wood bark, the microorganisms are attached by a mucous growth being formed as the microbes grow using sugar as the carbon source. The chlorophenol-degrading capacity of the bacteria Rhodococcus CP-2 and Rhodococcus chlorophenolicus PCP-1 is genetically stable but requires an induction. The contaminant- degrading capacity of the bacteria can be induced either prior to t *he immobilization or after the immobilization. In connection with the induction the bacteria are fed with the chemical to be degraded as well aε with the nutrient medium εo that the bacteria will produce the enzymeε which degrade the εpecific contaminant. In actual practice the biomaεε can be immobilized onto the εupport by circulating a pure culture εtrain incubated in a fer entor through the εupport maεε until a εufficient amount of biomaεε haε become attached to the εupport. The εupport σompriεing the immobilized chlorophenol-degrading microorganism of the genuε Rhodococcus is a novel product developed in connection with the present invention. Preferred εupportε in the embodimentε of the invention co priεe organic porouε materialε εuch as wood bark, wood chips and modified polyurethane resinε. The microorganiεms of the genuε Rhodo¬ coccuε which are immobilized on a support retain their activity for a long time. This is a great advantage in their commercial use. Since hydrochloric acid iε formed aε a product of the bio¬ degradation of chlorophenolε the reactor circulating liquid should be buffered during the degradation εtage. On the other hand no nutrients need be added to the reactor since the microorganisms in the reactor are capable of using the carbon deriving from the chlorophenol aε their εole carbon εource. The biomaεε of the reactor may need to be activated or re¬ generated after each or εeveral biodegradation εtageε by circulating nutrient-containing water in the reactor. The reactor is preferably fed with a sugar source favouring the growth of the microorganiεm in question. The bacteria are preferably fed with a carbon source which haε a maximum favourable effect on the deεired bacterial εtrain but which the minimum amount of other bacteria are capable of uεing. That way the deεired microbes increase in number and some detached biomass iε detected in the water discharged from the reactor. During the degradation and filtering stage, however, eεεentially no biomaεε will be detached from the εupport. After the regeneration or in connection with the regeneration it may be neceεsary or at least advantageous to re-induce the contaminant-degrading mechanism into the microorganiεm. This is done by conducting a small amount of the contaminant, e.g. chlorophenol into the reactor εo that the bioεyntheεis of the degrading enzyme is actuated. The reactor can be cleaned at need for example by waεhing with preεsurized water uεing the circulating pump or in the caεe of the reactor being clogged the cleaning may be performed through εuitable manholeε or charging holeε in the reactor. The preεent process is preferably implemented in a reactor syεtem having at leaεt two reactorε one of εaid reactors functioning in the filtering stage of the proceεs while the other one undergoes the biodegradation process. It may be advantageous to implement the present procesε in a syεtem having εeveral reaσtorε connected in parallel or in εerieε so that any one reactor may undergo the biodegradation process while the other reactors function as biofilters. In case the system comprises more than two reactors the treat¬ ment stages of the reactors are periodically circulated so £4 that the reactor which at a given time has been capturing contaminant for the longest time will be next connected to the degradation stage and the reactor which in the previous cycle underwent a degradation stage, and an eventual activation stage, is connected as the last one of a series of filtering reactors . Figure 1 illustrateε a reactor εuitable for the purification of chlorophenol-containing water. The bioreactor includeε εeveral departments 1 on top of each other, εaid departmentε being filled with a maεε functioning aε an attachment εurface for the microbes. The microbeε on the surface of said masε are capable of degrading chlorophenolε. Said maεs iε charged into the reactor through the charging openings 2. Said mass is placed on perforated trays 4 which εupport the filling masε and εeparate the reactor εpace into separate departmentε . Air is fed into the reactor through aerator unit 5 in caεe the degrading activity of the microbeε iε an aerobic procesε and requireε aeration. Water to be cleaned iε fed through pipe 10 by feed and circulating pump 7 through feed and circulation pipe 8 to the revolving εprinkler 6 which meters the contami¬ nated water uniformly into the reactor. At the top of the reactor there is an exhaust pipe 12 for any gas formed in the biodegradation and for the discharge of the aerating gas, and in the lower part of the reactor there iε a diεcharge pipe 11 for cleaned water. Each department compriεeε sample tapε 9 and the reactor further compriεeε a monitoring pipe indicated by the number 3 for detecting the liquid level. The circulation pipe 8 of the reactor further comprises thermal resistances (not shown) which can be uεed for heating the circulating water to the desired level required by the biodegrading activity of the microbes. Pipe 8 may further comprise a feed pipe which can be closed by a valve and which may be uεed for any feed of buffer and/or nutrient solution, which, nutrient solution specifically in the regeneration phase of the reactor is fed to the circulating water. In the reactor εyεtem there may be εeveral εuch reactors connected in series or parallel so that the water fed by the pump , (or pumps) can be directed to and from any reactor or away from the system by turning εuitable valveε . In εuch a εyste any reactor(s) may be shut off from the line of the feed water, whereby water may be circulated in εuch reactor(ε) respectively through the circulation pipe. The process according to the invention may be carried out also in a reactor according to Figure 2, wherein the support 1 comprises chlorophenol-degrading microbes attached to the surface thereof. The feed water is pasεed into the reactor through the inlet pipe 4 in the lower part of the reactor with the aid of feed and circulating pump 3. Having flowed through the biofilter 1 the cleaned water passes to a settling εpace 2 at the top of the reactor and the water iε further conducted away through diεcharge pipe 5. For the aerobic activity of the microbeε the reactor iε aerated through the perforated aerating pipe 6 in the lower part of the reactor, and the reactor gases are discharged through pipe 7. The following exampleε further illustrate the invention with¬ out, however, limiting it in any way. Example 1 Isolation of a contaminant-degrading microorganism 10 to 15 g sampleε of contaminated εoil or εlurry were in¬ oculated into 500 ml columns containing softwood bark chips as a solid εupport and 200 ml of mineral salts solution was percdlated through said columns. Tetrachloroguaiacol (TeCG) was added weekly to a concentration of 10 to 50 JUM. After three months it waε obεerved that the percolating fluids contained mixed cultureε which repeatedly removed added TeCG (10 uM) . The cultures obtained from the percolators where enridhed by repeated dilutions and by feeding with 10 to 20 JUM TeCG at 2 to 3 days' intervals. Samples from the cultures were streaked onto DSM-65 (glucose 4.0 g, yeast extract 4.0 g, malt extract 4.0 g, H2O 1000 ml, pH 7.2) agar containing 10 uM TeCG, and 130 colonieε from each mixed culture were teεted for TeCG-degradation by meaεuring the removal of TeCG from the εolution. The TeCG-degrading colonieε were purified. By a correεponding method uεing pentachlorophenol aε for enrichment εubεtrate Rhodococcuε CP-2 (DSM 4598) among otherε waε obtained. Said microorganiεm completely removed 10 uM pentachlorophenol (PCP) in five hourε and 70 % of the 1 C-labeled PCP evolved aε 14 C02 (see Fig. 3). Fig. 3 shows the degradation of 10 uM PCP spiked with 14 C PCP aε a function of time. The figure o in the curve designates the PCP concentration and the figure O designateε the evolution of 14 co 2 . The strain CP-2 also degraded other chlorinated phenols, guaiacols and εyringols in 10 uM solutions. In these tests pentachlorophenol (PCP), 2,3,4,5-tetrachlorophenol (2345- TeCP), 2,3,4,6-tetrachlorophenol (2346-TeCP) , 2,3,5,6-tetra- chlorophenol (2356-TeCP) , 2,3,4-trichlorophenol (234-TCP), 2,3,5-trichlorophenol (235-TCP), 2,3,6-trichlorophenol (236- TCP) , 3,4,5-trichlorophenol (245-TCP), 2,5-dichlorophenol (25-DCP), tetrachloroguaiacol (or tetrachloro-2-methoxyphenol) (TeCG), 2,4,5-trichloroguaiacol (345-TCG) , 3,4,6-trichloro- guaiacol (346-TCG) , 3,5,6-trichloroguaiacol (356-TCG), 4,5,6- trichloroguaiacol (456-TCG), 3,4-dichloroguaiacol (34-DCG), 3,5-dichloroguaiacol (35-DCG), 3,6-dichloroguaiacol (36-DCG) , trichlorosyringol (or trichloro-2,6-dimethoxyphenol) (TCS) and 3,5-dichloroεyringol (35-DCS) degraded in 48 hourε. By a corresponding method alεo bacteria degrading other con¬ taminants and other chlorophenol-degrading bacteria can be iεolated, which bacteria can be uεed in the proceεε of the preεent invention. Example 2 Immobilization of the microorganism onto a support Pure cultures of the chlorophenol-degrading microorganiεms Rhodococcus chlorophenolicus PCP-1 (DSM 43826) and Rhodococcus CP-2 DSM (4598) were immobilized on a polyurethane resin mass which comprised polyurethane resin REA 90/16 produced by Bayer Ag, West-Germany, which polyurethane was modified for the immobilization of bacteria (particle size below 10 mm; density from 1.14 to 1.05 kg/1 at 20 °C; weigth in εuεpenεion 115 kg as kg dry substances/m 3 volume of εuεpenεion; rate of sedi¬ mentation: 94 _+ 6 m/h) . The mixed culture of the bacteria waε incubated in a fermentor using 1 % glucose, 1 % sorbitol aε carbon εource in the preεenσe of the εupport. Aε they grew the bacteria excreted εlime and attached perceptively onto the εurface of the εupport material. When εufficient biomaεε had formed the in¬ cubation waε ended. The polyurethane mass containing the immobilized Rhodococcus culture was stored for 20 days at +4 °C. In a test performed after said storage the microbes were found to be still capable of degrading chlorophenolε. Chlorophenol-degrading Rhodococcus bacteria have been found to degrade chlorophenols even after having been stored at +4 °C for one year. Example 3 Bioreactor function The testε were performed in a 0.5 1 laboratory εize test reactor having a water feed opening and a gas exhaust opening at the top, a water discharged opening in the bottom, and an aeration opening in the lower part thereof. Modified poly¬ urethane REA 90/16 (producer Bayer AG, West-Germany) was used as support material. The amount of masε in the reactor was 400 ml. Rhodococcus chlorophenolicus (DSM 43826) bacteria and Rhodo¬ coccuε CP-2 (DSM 4598) were uεed aε degrading microbes. The microbeε were immobilized on the polyurethane reεin εupport by incubating them in a εuitable growth medium together with εaid resin. The teεt reactor waε filled with active reεin including both typeε of microorganiεmε in an immobilized form. An identical reference reactor (εterile) waε without microbes. The purpose of the sterile parallel teεt waε to check the binding of chlorophenolε onto the εurface of the masε and to eliminate the εhare of any non-biological mechaniεm in the degradation. The feed comprised chlorophenol-containing water having a chlorophenol content of either 0.1, 5, 10 or 20 mg/1. The feed rate was 2 vol/d. The temperature was +25 °C, the aeration rate 5 ml/min. A slight buffering waε performed with a phos- phate buffer for keeping the pH at about 7. In connection with the test chlorophenol degradation was observed: 1) by the increase of inorganic chloride in the water diε- charged from the biofilter. The chloride derived from chlorine bound to the chlorophenol molecule; 2) by adding 14 C-labeled pentachlorophenol into the reactor it was indicated that **- 4 C carbon dioxide deriving from the labeled PCP-ring waε discharged with the exhaust gas; 3) by measuring the total chlorophenol content of the dis¬ charged water and the fed water by gas chromatography; 4) by comparing the analyεiε reεultε from the active biofilter and the sterile biofilter it was noted that only the active column degraded chlorophenolε . Chlorophenols (in the form of a wood preservative called Ky-5, producer Kymi Oy, Kuusankoεki, Finland) were fed into the reactorε in accordance with the valueε in Table 1 during 63 dayε . The chlorophenol content waε measured in the feed solution by gaε chromatography. TABLE 1 The chlorophenol (Ky-5) content in the solution fed into the biofilterε during the test Chlorophenol content (mg/1) 3.4 6.2 12.8 20.3 0 25.3 0 25.3 0 The average chlorophenol content of the preservative Ky-5 is disclosed in percentages by weight in the following Table 2. TABLE 2 Chlorophenol Percentage 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 The nutrient concentration (N, P, etc.) waε very low in the liquid fed during the teεt εo that the cellε would not in¬ crease in number and the reactor leak microbeε. At the 7th day 14 C-labeled pentachlorophenol, having its ring carbon atoms randomly labeled, was fed into the reactors in order to indicate the mineralization of pentachlorophenol by 14 C-labeled carbon dioxide. During the whole test the sterile biofilter remained free of microbial growth. It did not discharge radio active carbon dioxide or inorganic chloride. The test resultε are εhown in Figε. 4 to 6. Fig. 4 εhowε the degradation in the biofilter of chlorophenol to inorganic chloride. The upper curve shows the sum of chlorophenol (CP) fed into the reactorε during the teεt and the lower curve εhows the produced chloride (Cl~) . The chloride forms about 60 % of the weight of the chlorophenol. A calculation on this baεiε indicateε that the immobilized cellε have degraded about 55 % of the added chlorophenol during the 63 days. The rest of the chlorophenol iε bound to the poly¬ urethane εupport. Fig. 5 εhows an indication of the biodegradation of penta¬ chlorophenol by the action of the immobilized bacteria. The radio active pentachlorophenol (the ring carbons randomly labeled by 14 C) (55500 cpm) added at the 7th day of the test decomposed into labeled carbon dioxide. The yield was nearly 50 %. A part of the labeled PCP remained out of range for the bacteria as it waε bound to the polyurethane maεs and a εmall amount waε possibly transformed to cell masε. No radio activity waε found to leak through the degrading reactor. Fig. 6 εhowε the amount of chlorophenol diεcharged from each of the reactorε . The εterile as well aε the bacteria- containing biofilter both effectively bind chlorophenols during 25 dayε . Thereafter the reactor wherein no degradation takes' place begins to leak chlorophenolε very quickly. The degrading biofilter, on the other hand, let through only very little chlorophenol, the content in the diεcharged water generally remaining below 10 /ug/1, which iε the accepted limit for the chlorophenol content in houεehold water. In the water diεctiarged from the εterile filter the content roεe up to a level of 1 mg/1 at the end of the 60 day teεt run, which iε a reεult of the polyurethane maεs being saturated with chloro¬ phenols. Example 4 Test performed with cold feed water The same test apparatus as the one in Example 5 was uεed in the test. Chlorophenol-containing cold (+5 °C) water was fed through the reactor during three days and a slight decreaεe in the chlorophenol binding capacity waε noted. Thereafter the reactor waε brought to room temperature (+25 °C) and it immediately εtarted to degrade chlorophenol. The amount of chloride in the circulated fluid increased heavily as an indication of chlorophenol degradation. During a continuos teεt (the teεt continued for 98 dayε) the purification of cold water waε εeveral ti eε teεted in the above deεcribed reactor. In theεe teεtε the reactor waε fed during 2 or 1 days water including chlorophenols in the amount indicated in Table 3. Then the feeding of the water waε dis¬ continued and water was circulated at +25 °C in the reactor for 5 and 16 days, respectively. The parameters of the test and the teεt reεultε are indicated in Table 3. The amountε of chlorideε in the liquid diεcharged from the teεt reactor and the reference reactor during the cold treat¬ ment were identical, but immediately when the reactors were heated the chloride content in the liquid discharged from the test reactor was higher than that from the reference reactor, i.e. the microbe activity starts immediately when the temperature riεeε to one favouring εaid microbial activity. In the foregoing the invention has been described mostly as a proceεs for removing chlorophenols. For a person skilled in the art it iε, however, clear that the corresponding method can be utilized also for other biodegradable contaminants when εuch microorganiεmε degrading εuch contaminantε can be attached to a biofilter capable of capturing εaid contaminant. ■ ? TABLE 3 ∞ E Absorption of chlorophenols in cold with subsequent degradation at +25 °C Test day 99 2/5 49 84 3335 21 00 ( 105 2/5 81 94 4081 10 20 110 1/5 100 28 3104 15 117 2/16 284 31 1203 53 31 O

Claims: Claims 1. A procesε for the microbiological purification of polluted water with the aid of contaminant-degrading microorganiεmε, in which proceεε the water to be purified iε directed through a biofilter and the microorganiεms immobilized onto a biofilter are made to degrade the εaid contaminant, c h a r a c t e ¬ r i z e d in that the purification iε performed in an eεsentially two-stage proceεε, wherein water to be cleaned iε fed in a firεt or filtering εtage through a biofilter capable of capturing the contaminant, and in a second or bio¬ degradation εtage the flow of the feed water through the bio¬ filter iε diεcontinued and the microorganiεmε immobilized on the biofilter are made to degrade the captured contaminants. 2. A procesε according to claim 1, c h a r a c t e r i z e d in that the feeding of the water to be purified into the bio¬ filter iε diεcontinued before the contaminant concentration in the filter has risen above the tolerance level of the im¬ mobilized microorganiεmε and/or the saturation point of the biofilter. 3. A proceεε according to claim 1 or 2, c h a r a c t e ¬ r i z e d in i:hat a relatively small amount of water εurrounding the biofilter iε circulated in εaid biofilter during the biodegradation stage. 4. A process according to claim 1, 2 or 3, c h a r a c t e ¬ r i z e d in that the parameters of the water εurrounding the biofilter during the biodegradation εtage are adjuεted in order to obtain and/or retain an environment which is favourable for the activity of the degrading microorganisms. 5. A procesε according to claim 4, c h a r a c t e r i z e d in that the parameters to be adjusted are chosen from the group consisting of the water temperature, the pH-value, the nutrientε, etc. 6. A proσeεε according to any one of the preceding claims 1 to 5, c h a r a c t e r i z e d in that the water to be purified, such as εurface or ground water, is colder than the temperature of activity of the degrading microorganismε, and in that the temperature during the filtering εtage iε kept at the temperature of the feed water, and that the temperature of the water εurrounding the biofilter is raised at the beginning of the biodegradation εtage to a level which iε favourable to the activity of the degrading microorganisms, generally to about 20 to 35 °C, and that said water is retained at εaid temperature until the captured contaminantε have eεεentially decompoεed. 7. A proceεs according to any one of the preceding claims 1 to 6, c h a r a c t e r i z e d in that the contaminant com¬ prises chlorinated phenolic compounds and/or their derivatives. 8. A proσeεs according to claim 7, c h a r a c t e r i z e d in that the degrading microorganismε comprise bacteria of the genus Rhodococcus which degrade chlorinated phenolic compounds and their derivativeε. 9. A proceεε according to claim 8, c h a r a c t e r i z e d in that the bacteria are Rhodococcuε εp. CP-2 (DSM 4598) and/or Rhodococcuε chlorophenolicus PCP-1 (DSM 43826) . 10. A procesε according to any one of the preceding claimε 1 to 9, c h a r a c t e r i z e d in that the biofilter com¬ priεeε a porous material which is capable of reverεibly capturing chlorinated phenolic compoundε and their derivatives and which iε capable of functioning aε an attachment εubεtrate for εaid bacteria, preferably a polyurethane reεin, wood bark or wood chipε. 11. A proσesε according to claim 10, c h a r a c t e ¬ r i z e d in that the biofilter compriεeε a polyurethane reεin which iε modified for the immobilization of microbeε and to which chlorophenol-degrading Rhodococcuε bacterial cultureε are immobilized. 12. A proceεε according to any one of the preceding claimε 1 to 11, c h a r a c t e r i z e d in that when the biofilter has gone through the biodegradation stage it is transferred back to the filtering εtage by directing thereto water to be purified, and that this chain of activity composed of said first and said second stages, is repeated. 13. A process according to any one of the preceding claims 1 to 12, c h a r a c t e r i z e d in that the biofilter com¬ prises two or several biofilterε connected in εeries or parallel so that while one biofilter is functioning in the biodegradation εtage the other biofilterε filter contaminantε from the feed water. 14. A process according to claim 13, c h a r a c t e ¬ r i z e d in that feed water is directed through two or several biofilters connected in series and that the biofilter which was first in the flow sequence iε tranεferred to the biodegradation εtage and the biofilter which waε functioning in the biodegradation εtage is connected as the laεt filter in the feed water flow εequence. 15. A proceεε according to any one of the preceding claimε 1 to 14, c h a r a c t e r i z e d in that the microorganiεms immobilized on the biofilter are regenerated at intervals after a performed biodegradation εtage by circulating nutrient-rich water through εaid biofilter εo that the micro¬ organiεmε increaεe in number. 16. A pure culture of a microorganiεm capable of degrading chlorinated phenolic compoundε and their derivatives, c h a r a c t e r i z e d in that said microorganism is Rhodococcus εp. CP-2, DSM 4598, which haε been induced to degrade chlorinated phenolic compounds and their derivativeε. 16. A solid porous εupport material compriεing immobilized microorganiεmε capable of degrading chlorinated phenolic com¬ poundε and their derivatives, c h a r a c t e r i z e d in that said support material comprises a polyurethane reεin having a large εurface area and the capacity of reverεibly capturing chlorinated phenolic compoundε and/or their derivatives, said support material having immobilized thereon a mixed or pure culture or cultures of said degrading micro¬ organisms. 17. Α support material according to claim 16, c h a r a c ¬ t e r i z e d in that said support material compriseε a poly¬ urethane resin modified for the immobilization of bacteria and in that the microorganism compriseε a culture or cultures of the genus Rhodococcus. 18. A support material according to claim 17, c h a r a c ¬ t e r i z e d in that the microorganism is Rhodococcus chlorophenolicus PCP-1 (DSM 43826) and/or Rhodococcus sp. CP-2 (DSM 4598) .

Inventors: HAEGGBLOM MAX MIKAEL, VALO RISTO JUHANI, HAEGGBLOM, MAX, MIKAEL, VALO, RISTO, JUHANI
Applicants: ALKO LTD
Application year: 1989
Main classification: C02F3
IPC codes: C02F3, C12N1, C12N11, C12R1