Title of Invention

PROCESS FOR SELECTING BACTERIA SUITABLE FOR OXIDIZING ARSENIC.

Abstract

TITLE: PROCESS FOR SELECTING BACTERIA SUITABLE FOR OXIDIZING ARSENIC. There is disclosed a process for selecting bacteria suitable for oxidizing arsenic in the form of As(III) into arsenate As(V) using CO2 as the sole source of carbon and the As (III) as the sole source of energy, said bacteria presenting in addition at least one of the following characteristics: - they withstand up to at least 1 g.1-1 of As(III) without losing their ability for oxidizing the As(III), - the oxidation rate of the As(III) by these bacteria, in a bioreactor, reaches at least 4 mg.1-1.h-1, As(III) concentration beign comprised between 100 and 1000mg.1-1 and bacteria concentration being 106 bacteria per ml, - their specific activity of ixidising the As(III) reaches at least 5x10-7mg of As (III) per bacterium per hour. - they are capable of reducing the residual concentration of As(III) to less than 50ug.1-1 said process comprising the steps of : (a) placement of a sample capable of containing such bacteria in contact with a selection medium containing from 50 to 500 mg. 1-1 of arsenite As(III) and mineral salts; (b) placement of the sample and the selection medium in a culture in sterile conditions, in pH conditions comprised between 5.5 and 6.5 and of conditions of temperature comprised between 20 degree C and 35 degree C. (c) selection of suitable bacteria for oxidizing the As(III) into As(V), if necessary by successive sub-samplings in the selection medium.

Full Text

PROCESS FOR SELECTING BACTERIA SUITABLE FOR OXIDIZING ARSENIC The present invention relates to process for selecting bacteria suitable for oxidizing arsenic, and in particular, to oxidize arsenic in the form of arsenite As(III) into arsenate As (V). It also applies to a process for the selection of such bacteria, as well as their applications for treating arsenic in the form of As(III). The presence of arsenic can be observed in natural media, as in materials and residues resulting from human activities. It is thus found in underground water, and originates in the geo- chemical bottom. It is also detectable, for example, in residual, industrial, waste-water effluents, mineral-processing residues, coal combustion, waste- treatment, deposits of industrial residues, or when fertilizers are used. Various methods of eliminating arsenic were proposed, given that the valency under which arsenic emerges from processing is very substantial in elimination outputs. Thus, the best eliminated form corresponds to the As(V) in the following treatments: co-precipitation by iron salts or by aluminium salts, or during decarburisation with lime, and reverse osmosis. Another method of elimination is based on absorption in columns. The most frequently-used material is constituted of activated aluminium, which allows good retention of arsenic (III) and arsenic (V). However, the regeneration of the column necessitates the use of concentrated caustic soda. It ensues that the recovery of the As(III) is two times less high than that of the As(V). Other techniques, which use ion-exchange resins or nanofiltration are complicated to use and involve high investment and maintenance costs. In addition, the nanofiltration and reverse osmosis involves the loss of apart of the water in the form of an arsenic-concentrated effluent. However, a recent study carried out by the "Geological Survey of Finland" has shown that the three methods currently proposed for the elimination of arsenic in water emanating from private drillings, were not efficient in the long term. These methods correspond to the use of cartridges with activated aluminium, activated-carbon cartridges, or ion-exchange resins. The best results were obtained by oxidizing As(III) into As(V) and then combining two methods of purification. As stressed in literature, the efficiency of most of these treatments depends on the stage of oxidation of the As(III) into As(V). The oxidation of As(III) may be carried out by chemical means, with, for example, chloride, ferric chlorine, potassium permanganate or ozone. The works of inventors in this field have shown that, in an unexpected way, such an oxidation process could be carried out by biological catalysis. It therefore appeared that it was possible to economise on chemical-oxidation reagents by using bacteria selected for their specific oxidizing properties, enabling As(III) to be oxidized into As(V). The object therefore of the invention is to supply novel bacteria capable of oxidizing As(III) into As(V), less toxic, less mobile and therefore easier to eliminate by conventional physico-chemical treatments of the treated media. According to another aspect, the invention relates to a process for the selection of such bacteria, as well as selection media as novel products. According again to another aspect, the invention also relates to the use of novel bacteria for the treatment of media containing arsenic. The bacteria according to the invention are characterised in that they are isolated, autotrophic, aerobic bacteria, capable of oxidizing As(III) into As(V), using CO2 as the sole source of carbon and As(III) as the sole source of energy, and of which the 16S ribosomal DNA presents at least 95% identity with SEQ ID no. 1 or SEQ ID no. 2. However, at least the part of the DNA of the bacteria according to the invention conferring the said properties on them, is capable of hybridising with the genome of the phylum filed with CNCM 28, rue du Dr Roux Paris, France, on 20 June 2001, under no. I-2687. It indeed appeared that the bacteria according to the invention emanated from a selection process as described hereinafter for the most part fall within a single species. This observation ensues from a sequencing of the region of the DNA encoding for the 16S ribosomal DNA, hereinafter called 16S rDNA, after amplification of the region of the genome of these bacteria, corresponding to region 18-1492 of the genome of E.coli, of which the accession number is cited by the European Molecular Biology Library (EMBL) under number EMBL J01195, in accordance with the method provided in Brosius et al, 1981, Journal of Molecular Biology, 148: 107-127. This amplified 16S rDNA was cloned in E. coli, then sequenced. The rDNA sequences thus obtained, carried out on 54 different clones indicate a divergence rate in excess of 5 %, with all the bacterial species listed, and a divergence of less than 3 % between the various, selected clones analysed, as explained in detail in Example 1, hereinafter. The sequences ID no. 1 and no. 2 represent the 16S rDNA emanating from clones C02 and C19 obtained from sample CAsOl; the phylum CAsOl containing the sequence ID no. 2 was filed with the CNCM on 20 June 2001 under number 1-2687. The bacteria of the invention present moreover at least one of the following characteristics: - they withstand up to at least 1 g. 1-1 of As(III) without losing their ability for oxidizing the As(III), - the oxidation rate of the As(III) by these bacteria in a bioreactor reaches at least 4 mg. 1-1.h-1, - their specific activity of oxidizing the As(III) reaches at least 5x10- 7 mg of As(III) per bacterium, per hour. With such a specific activity value, a bacterial suspension containing 107 bacteria per ml is capable of oxidizing 5 mg of As(III) per litre per hour. It ensues that the oxidation of the As(III) by the bacteria, according to the invention, necessitates only a very low production of bacterial organic matter. Consequently, the quantities of nutriments to supply for the development of the bacteria (nitrogen, phosphorous) are also very low. - they are capable of reducing the residual concentration of As(III) to less than 50 µ g.l-1. The bacteria according to the invention and presenting the characteristics hereinabove are comprised within a group constituted of: a) the phylum filed with the CNCM on 20 June 2001 under number I-2687 and named CAsO1 hereinafter; b) phyla of which the genome presents 80 % identity, preferably 90% identity, with the phylum in a), the identity being understood by a capacity for identical transcription and translation. The invention also relates to a process of selection and isolation of the bacteria described hereinabove. This process is characterised in that it comprises the contacting of a sample susceptible of containing such bacteria, in particular taken from a material contaminated by arsenic, with a selection medium containing a ratio of 50 to 500 mg. 1-1 of arsenic in the form of arsenite, not containing an additional source of carbon and supplying solely the As(III) as a source of energy for the bacteria. The sample and the selection medium are placed in conditions of sterile and aerobic culture. The material containing the bacteria which it is desired to select, represents 5 to 20% of the final culture mass The incubation temperature is comprised between 20 and 35°C, and preferably in the region of 25°C and the pH comprised between 5.5 and 6.5. The contaminated material originates, for example, from a mining or industrial site. In the course of the culture, dosages of As(III) and As(V) are carried out each day. The culture is sub-sampled when all the As(III) has been oxidized. Given that the selection medium is inoculated with a sample capable of containing compounds able to permit the growth of undesirable bacteria, it is preferable to carry out successive sub-samplings, in order to dilute the undesirable compounds. Sub-samplings at approximately 10% are advantageously carried out about every four weeks for the first 3 sub- samplings, then each week for the following. The selection medium gradually enables the proportion of the autotrophic bacteria oxidizing As(III) into As(V) to increase in relation to the proportion of the other bacteria present or undesirable. Moreover, the absence of additional sources of carbon also provide the development of the sole autotrophic bacteria capable of developing, using the atmospheric CO2 as a source of carbon. In short, the absenec of source of energy other than the As(III) contributes to the elimination of the undesirable bacteria. Such a selection medium is novel and, as such, also enters into the field of the invention. It is also usable for the conservation of the bacteria. For the growth of the bacteria, apart from As(III), with a concentration comprised between 50 and 500 mg.l-1, this medium contains to advantage mineral salts and/or trace elements and/or vitamins in an aqueous solution, to advantageous effect, it being understood that these nutriments must not constitute a pool of oxidizable compounds which might be used by undesirable bacteria as a source of energy. The mineral elements comprise to advantage nitrogen, phosphorous, potassium, magnesium, calcium, sodium and a chloride. An appropriate medium for the selection of the bacteria according to the invention is a medium buffered with a pH substantially equal to 6 and containing : - arsenite As(III) with a concentration of 50 to 500 mg 1-1; - ammonium = 14 mg.l-1, representing 10 mg.l-1 of nitrogen; - mineral salts providing a nutriment function and/or the buffering effect mentioned; The following mineral salts may be: - Phosphates of di- and mono- potassium, which also serve as a pH buffer comprised between 0.05 and 2 g. 1-1 - A non-biologically oxidizable magnesium, such as the sulphate of magnesium between 0.05 and 1 g. 1-1 - A non-biologically oxidizable calcium, such as the calcium chloride between 0.05 and 1 g. 1-1 - Sodium and chloride, notably in the form of sodium chloride or another non-biologically oxidizable salt between 0.05 and lg. 1-1. - Metals: (trace elements): traces It was observed that the growth of the bacteria corresponding to the invention is correlated with the oxidation rate of the As(III), as shown in the examples. The bacteria able to be selected by the process hereinabove, using the selection medium as defined, also form a part of the invention. The invention also concerns a process for treating media containing arsenic to oxidize the arsenites into arsenates characterised by the inoculation of these media in a bioreactor, with a bacterial population essentially constituted of bacteria such as those defined and selected above. It is advantageous to work in an aqueous environment. A bacterial culture is used to seed a medium to be treated in such a way that the final concentration of bacteria are comprised between 5X105 and 5x106 bacteria per ml, and preferably approximately 106 bacteria per ml. In a discontinuous culture, or a batch, that can be obtained by inoculating the medium for treatment with 10 % volume in the volume of a bacterial culture at confluence, i.e. approximately 107 bacteria per ml. In another embodiment, the bacteria may be at least partly concentrated in the bioreactor with the mean of a support on which the bacteria may adhere. When it is desired to accelerate the oxidation, mineral salts serving as nutritive elements are added to the aqueous solution and/or the air is injected when the oxygen concentration in the medium to be treated is limiting (which depends on the initial As(III) concentration). The mineral elements are used in a ratio of approximately 0.1 to 0.5 g.l-1, notably of 0.01 to 0.1 g.l-1 of the inoculated medium and treated. It is advantageous for these elements to be chosen from among nitrogen, phosphorous, potassium and magnesium. It is advantageous for the treatment process to be carried out with a pH comprised between 5.5 and 6.5, preferably in the vicinity of 6, and at a temperature comprised between 20 and 35°C, preferably between 24 and 26°C. The microbiological treatment is started in batch mode: the As(III) contained in the solution to be treated is oxidized by the bacteria. The bioreactor can then be supplied by a medium to be treated continuously. Mineral elements as indicated hereinabove will, if necessary, be added to this medium. It is advantageous for the work to be carried out in conditions enabling a homogenous and effective contact of the aqueous medium with the bacteria. To this effect, the reactional medium is subjected, for example, to agitation, using a bioreactor fitted with an agitation device. As a variant, a column bioreactor with a fixed bacterial bed is used. The use of this process enables the toxicity of the medium to be reduced, leading to residual concentrations of As(III) of less than 50 µg.l-1, as required by the European Directive dated 3 November 1998, relating to the limit concentration value of arsenic in water intended for human consumption. The arsenates formed can easily be eliminated because their mobility is lower than that of the arsenites. The solution recovered from the bioreactor which mainly contains arsenic in the form of As(V) and a residual concentration of As(III) lower than its initial concentration in the medium treated, is directed to a device for the elimination of the As(V). This device permits, for example, a co- precipitation to be effected by iron salts or aluminium salts or a co- precipitation during treatments of de-carburisation with lime, a reverse osmosis, an absorption into columns, or any other process or combination of processes leading to the elimination of the As(V). The process of the invention is, in particular, usable to treat media rich in arsenic, such as water intended for human consumption, communal supply or private drillings, as well as the effluents, whether mining or industrial, or underground water, notably in a reactive-barrier, "in situ" process. Although not limitative, described hereinafter, by way of examples, will be various embodiments of the present invention, having reference to the accompanying drawings, in which : Figure 1 is a graph showing the variation in the concentration of As (V) as a function of the bacteria batch-oxidation time constituting the CAsOl population. Figure 2 is a graph showing the variation in the oxidation rate of the As(III) as a function of the time in a continuously-functioning bioreactor of bacteria constituting the population of the CAsOl. Figure 3 is a graph showing, as a function of the time, respectively, the time of residence of the bacteria in the reactor and the oxidation rate of the As(III). Figure 4 is a graph showing the variation in the oxidation rate of the As(III) as a function of the pH of the medium. Figure 5 is a graph showing the influence of the oxygen concentration on the oxidation rate of the As(III) by the bacteria constituting the bacterial concentration CAsO l. Figure 6 is a graph showing the influence of the temperature on the oxidation rate of the As(III) by the bacteria constituting the CAsOl population. Example 1: molecular identification of a bacterial population used for the treatment of arsenic - Techniques used Principle of the molecular inventory The identification effected is based on an analysis of the RNA 16S of the bacterial ribosomal sub-unit, and of the 16 S rDNA sequence of which it is transcribed, as a basis of comparison with other bacteria. It should be borne in mind that it is allowed in this regard for a divergence of 3% corresponds to the phenotypic distinction between two species. After amplification, the cloning in E-coli of the 16S rDNA of each bacterium present in the sample is initiated, then the sequencing, a partial sequencing of 500 pb in all cases, and complete sequencing of 1500 pb if the diversity is narrow. The sequences obtained are firstly compared among themselves using a computer software to distinguish each species present on the basis of the percentage divergence. An interrogation of databanks then enables the micro-organisms most closely-related to the sequences representing each species listed to be inventoried. Genbank and Ribosomal Data Project were therefore interrogated. The sequences representing the species inventoried were once again compared with those of the related micro-organisms, on the basis of the percentage of divergence. SSCP Analysis The SSCP technique (Single Strand Conformation Polymorphism) is based on the secondary conformation which a fragment of single-strand DNA adopts in non-denaturing conditions (thanks mainly to Van der Waals type attractions) and which is dependent on its primary structure (sequence). A particular region of the 16S rDNA (region V3 situated . approximately between positions 300 and 500) of the micro-organisms present, is amplified, denatured to separate the DNA strands, then analysed by electrophoresis on electrophoresis gel in non-denaturing conditions. The SSCP analysis profile of the total population enables its complexity to be determined and each micro-organism present to be differentiated, on account of the migration speed of its rDNA fragment. The assignment of each peak is then carried out by comparison with the individual, SSCP-analysis profiles of each clone representing the species registered during the molecular inventory. Results - stabilization of the samples From a sample of 15 ml, containing approximately 2x109 bacteria/ml, 7 ml of sample is taken, which is immediately centrifuged and the residue is absorbed in 250 µl of guanidine thiocyanate and 10% of 37.5 µl of N- lauryl sarcosine. The sample thus stabilised is preserved at -20°C until the total DNA- extraction stage. - analysis and inventory of the bacterial population. The DNA extracted from the sample was subjected to an amplification reaction of the V3 region of the 16S rDNA present, and was analysed according to the SSCP technique. The SSCP electrophoregram showed that one species is essentially the majority one, since a single peak appears on the SSCP profile of the total population. Subsequent analysis of the sequences of the clones for the inventory of the bacterial population, as well as the individual SSCP analyses of clones confirm this observation. To carry out the inventory, a library of 54 clones was constituted each containing a bacterial 16S rDNA fragment. In order to differentiate the various populations present, a screening was carried out by means of a digestion with the restriction enzyme Hae III of the 16S rDNA fragment of each clone, followed by an electrophoresis on agarose gel. The 54 clones analysed showed an identical digestion profile. This result confirms the heavy-majority presence of a single species, such that solely the rDNA of this species is amplified and is detectable. A search in two databanks indicated that the majority population, exclusively detected by the techniques hereinabove, from a phylogenetic viewpoint, does not belong to any species already known and listed on the databanks. Indeed, the most closely-related species is Thiomonas thermosulfata (Proteobacteria; subdivision Beta; Thiomonas) with a relatively high percentage of divergence, since comprised between 7.9 and 7.3% in the best of cases (Table 1). Table 1. Clones, the 16S rDNA of which was sequenced and the percentage divergence in terms of the most closely-related organism from a phylogenetic viewpoint, which is Thiomonas Thermosulfata. *: a divergence of less than 3% corresponding to the same species. - An individual SSCP analysis of the clones and assignment An SSCP analysis was carried out on 4 clones taken at random, in view, on the one hand, of the simplicity of the SSCP profile of the total population, and on the other hand of the perfect preservation of region V3 of the 16S rDNA of the clones of which the sequence was analysed. The SSCP profile of the 4 clones analysed was exactly superimposed on the peak of the SSCP profile of the total population thus conclusively confirming the exclusive detection of one species of bacterium. - complete analyses of the 16S rDNA sequences. In view of the substantial divergence of the first 500 nucleotides of the 16S rDNA sequence of the species detected in the sample (3.7. to 7.9%) with the sequences listed in the databanks, an analysis of the complete sequence of the 16S rDNA of the 2 groups of main sequences listed, i.e. groups C6 and C2 was carried out. Although the percentage of divergence in the first 500 nucleotides of the 16S rDNA is considered to be representative of the totality of the sequence, this additional data will thus enable the phylogenetic originality of the species detected to be confirmed. The analysis of the 16S rDNA of the C02 clones (group C2) and C19 (group C6) shows on the one hand that the sequences are divergent from one another by only 0.1% and on the other hand that after comparison with the databanks these sequences belong indeed to a different species from those currently known and listed (Table 2). Table 2. Percentage divergence of the 16S rDNA of the C02 and C19 clones with those of the most closely-related micro-organisms**. **: a divergence of less than 3% corresponds to the same species. The 16S rDNA sequences corresponding to the C02 and C19 clones are supplied respectively in the ID No. 1 and ID No. 2 sequences. GenBank indicates that the most closely-listed sequence is that of a beta Proteobacterium of the Comamonadaceae family called dehydroabietic acid-degrading bacterium DhA-7. The Ribosomal Data Project databank indicates that the most closely- related sequences are those of Leptothrix discophora str. SS-1 and Leptothrix mobilis str. Feox-1 also of the Comamonadaceae. The molecular inventory study carried out on the population of the CAsOl sample thus shows that a single population was detected and the micro-organisms that are the most closely related from a phylogenetic viewpoint are the beta Proteobacteria Thiomonas thermosulfata, Dehydroabietic acid-degrading bacterium DhA- 71,Leptothrix discophora str. SS-1 and Leptothrix mobilis str. Feox-1. The detection of a single species of micro-organism in the CAsOl sample indicates that the medium of the sample is extremely selective and close to the conditions of a pure culture (no other contaminating species was detected among the 54 clones analysed). The comparative works show that the population of CAsOl is novel, since no known bacterium listed in the databanks possesses a divergence lower than at least 6.2 % with the organism inventoried. Example 2: Composition of a selection medium I — Preparation of the culture medium • Base medium The base medium is composed of a solution A and a solution B. Solution A: Solution A is prepared in a glass flask of 1 litre. The pH of the solution is adjusted to pH 6. Solution B : Solution B is prepared in a glass flask of 500 ml. Solutions A and B are sterilised by autoclaving for 20 minutes at 120°C. • Solution of As(III) An aqueous solution of As2O3 of 13 g.l-1, corresponding to a solution of 10 g.l-1 of As(III) is prepared in the following way. As AS2O3 dissolves little in water, it is necessary to initiate a warm dissolution in a basic medium (0.55 g of NaOH in 100 ml of boiling water). The solution of As(III) is poured in a 1 litre sterile glass flask. • Solution of trace-elements The trace-element solution is sterilised by filtration at 0.45 µm in a sterile glass flask. • Solution of vitamins The vitamin solution is sterilised by filtration at 0.45 µm. II - Preparation of the complete medium In sterile conditions, it is necessary to add to the media the following solutions. The medium is stored at ambient temperature. Example 3: Batch oxidation process of As(III) by bacteria constituting the CAsO1 population The CAsOl bacterial population, selected with the medium in example 2, is cultivated in a medium containing 100 mg.l-1 of As(III). The experiments are carried out in sterile Erlenmeyer glass flasks of 500 ml, placed on an agitation table incited by a "to and fro" motion, thermostatically-regulated at a temperature of 25°C. These Erlenmeyer flasks containing 250 ml of the medium in example 2, are inoculated with 10% of CAsOl bacterial suspension at a concentration of 10 7bacteria ml-1. Without a previous adaptation phase, this bacterial population is used to inoculate media containing between 100 and 1000 mg. 1-1 of As(III). The oxidation kinetics of the As(III) into As(V) thus obtained are shown in the graph of figure 1, where the axis of the ordinates shows the concentration of As(V) generated by the oxidation of the As(III), of mg. 1-1, and the axis of the abscissa shows the time in hours. When the medium is initially adjusted to pH 6, the oxidation of the As(III) into As(V) is correlated with a drop in pH. This example shows that the CAsOl bacterial population keeps its capacity to oxidize the As(III) when it is placed in a solution containing 1000 mg.l-1 of As(III), i.e. ten times the concentration of As(III) present in its usual culture medium. Example 4: Continuous process of oxidation of the As(III) by the bacteria constituting the CAsOl population The CAsOl bacterial population, selected with the medium of example 2, was used to inoculate a reactor provided with an agitation shaft fitted with a screw, and a system of air supply. This bioreactor was filled with a solution containing 100 mg. 1-1 of As(III). After the batch phase, during which the bacteria entirely oxidized the As(III) into As(V), the bio- reactor was continuously supplied with a solution containing 100 mg. 1-1 of As(III). The supply flow of the solution charged with arsenic was gradually increased. The time of residence of the solution in the bioreactor was thus reduced from 150 hours to 10 hours. The percentage oxidation of the arsenite by the bioreactor was always greater than 99%. The increase in the rate of the oxidation of the As(III) in the bioreactor is shown in the graph of figure 2, where the axis of the ordinates represents the rate of the oxidation of the As (III) of mg. l-1.h-1, and the axis of the abscissa represent the time in hours. It shows that the oxidation rate may be 12 mg AS(III). l-1.h-1, while in a batch, this rate is a maximum of 4 mg. l-1.h-1. Example 5: Continuous process of oxidation of the As(III) in a bioreactor with a bed fixed by the bacteria constituting the CAsO1 population The bacterial population was used to inoculate a fixed-bed column reactor, containing pozzolana as the mineral support on which the bacteria may adhere. An injection of air at the base of the column enabled oxidization of the system to be supplied. The bioreactor was continuously supplied with a culture medium containing 100 mg.l-1 of As(III). The nutriments were supplied in the following proportions: 50 mg.l-1 of (NH4) 2SO4, 50 mg. 1-1 of MgSO4, 7 H2O, 50 mg. 1-1 of K2HPO4, 50 mg.l-1 of KH2PO4, 50 mg. 1-1 of NaCl. It was not necessary to add a source of calcium to this medium, as the latter was prepared with tap water already containing calcium. The treatment was carried out in non-sterile conditions. The supply flow of the solution charged with arsenic was gradually increased. The time during which the solution resided in the bioreactor was thus reduced from 300 hours to 2 hours. The percentage of oxidation of the arsenic (III) by the bioreactor was always greater than 95%. The increase in the oxidation rate of the As(III) by the bioreactor and the diminution in the time of residence are shown in the graph 3 of figure 3, in which the axis of the left ordinates represent the time of residence in hours, the axis of the right ordinates represent the oxidation rate of the As(III) at mg. 1-1.h-1 and the axis of the abscissa represents the time in days. In such conditions, it was possible to obtain a rate of oxidation at least equal to 38 mg. 1-1 h-1. Example 6: Influence of the pH on the oxidation rate of the As(III) by bacteria constituting the CAsO1 population The CAsOl bacterial population was used to inoculate reactors of which the usable volume was of 1 litre, and which was equipped with means of agitation constituted of a shaft fitted with a screw, and a system of air supply. These reactors were filled with one litre of mineral medium containing 100 mg.l-1 of As(III). The initial pH of the medium in these reactors was adjusted to different values comprised between 2 and 10, as shown in figure 4. The reactors were inoculated with 100 ml of CAsOl culture. The cultures were produced in so-called "batch" condition, which means that the medium has not been renewed. Frequent samplings were done in the reactors and enabled the process of the oxidation of the As(III) to be monitored, and the oxidation rate of the latter to be computed for each pH condition. The influence of the pH on the oxidation rate of the As(III) by the CAsOl bacterial population is therefore illustrated in figure 4, and it is observed in the latter that this rate is maximum at pH between pH 5 and pH 7. It should also be noted that the rate is still high between pH 3 and pH 8, but becomes low for pH values of less than 3 and greater than 8. Example 7: Influence of the concentration of oxygen on the oxidation rate of the As(III) by bacteria constituting the CAsO1 bacterial population This CAsOl bacterial population was used to inoculate a reactor with a usable volume of 10 litres, equipped with agitation means constituted of a shaft fitted with a screw and a system to enable injection of nitrogen or air into the culture medium. The reactor was filled with 10 litres of mineral medium containing 100 mg.l-1 of As(III). The initial pH of the medium in this reactor was adjusted to 6. The air present in this reactor was entirely chased by a flow of nitrogen, then the concentration of oxygen in the medium was adjusted to different values comprised between 0 and 0.3 mMole 1-1 by injection of low volumes of air. The reactor was inoculated with one litre of CAsOl culture. Successive tests with different concentrations of oxygen were carried out, as previously, in batch conditions. Frequent samplings carried out in the reactor enabled the process of the oxidation of the As(III) to be monitored and the oxidation rate of the latter to be computed for each oxygen concentration. The influence of the oxygen concentration on the oxidation rate of the As(III) by the CAsO1 bacterial population is illustrated in figure 5. This maximum oxidation rate was obtained as soon as the initial dissolved- oxygen concentration reached 0.05 mMole. 1-1, i.e. approximately 10% of the concentration when the liquid medium was saturated with oxygen. In these conditions, it was observed that the oxygen requirement of the CAsO1 bacterial population to oxidize the As(III) is law. Example: Influence of the temperature on the oxidation rate of the As (III) by bacteria constituting the CAsOl bacterial population As well as previously, the CAsOl bacterial population was used to inoculate reactors of which the usable volume was this time one litre, and which was equipped with means of agitation and a system of air supply. The reactors were filled with one litre of a mineral medium containing 100 mg. 1-1 of As(III). The initial pH of the medium in these reactors was adjusted to 6. The reactors were thermostatically-regulated reactors by the circulation of water emanating from a cryothermostat in a double shell. The temperature was maintained at different fixed values comprised between 10°C and 35°C. The reactors were inoculated with 100 ml of CAsOl culture. These cultures were produced in batch condition as previously. Frequent samplings executed in the reactors enabled the process of the oxidation of the As(III) to be monitored, and the oxidation rate of the As (III) to be computed for each temperature condition. The influence of the temperature on the oxidation of the As(III) is illustrated in figure 6. It was observed in the latter that the oxidation rate of the As(III) by the CAsOl bacterial population is the maximum from a temperature of 20°C, and that this oxidation rate is still high at temperatures of 20°C, and even 15°C, but that it is substantially lower at a temperature of 10°C. WE CLAIM: 1.- A process for selecting bacteria suitable for oxidizing arsenic in the form of As(III) into arsenate As(V) using CO2 as the sole source of carbon and the As(III) as the sole source of energy, said bacteria presenting in addition at least one of the following characteristics: - they withstand up to at least 1 g.l-1 of As (III) without losing their ability for oxidizing the As (III), - the oxidation rate of the As (III) by these bacteria, in a bioreactor, reaches at least 4 mg.1-1.h-1, As (III) concentration being comprised between 100 and 1000 mg.l-1 and bacteria concentration being 106 bacteria per ml, - their specific activity of oxidising the As(III) reaches at least 5x10- 7 mg of As(III) per bacterium per hour. - they are capable of reducing the residual concentration of As(III) to less than 50 µg.l-1, said process comprising the steps of: a) placement of a sample capable of containing such bacteria in contact with a selection medium containing from 50 to 500 mg. 1-1 of arsenite As(III) and mineral salts; b) placement of the sample and the selection medium in a culture in sterile conditions, in pH conditions comprised between 5.5 and 6.5 and of conditions of temperature comprised between 20°C and 35°C; c) selection of suitable bacteria for oxidizing the As(III) into As (V), if necessary by successive sub-samplings in the selection medium. 2.- A process as claimed in claim 1, in which the material containing the bacteria that it is desired to select represents 5 to 20% of the final mass of culture. 3.- A process as claimed in claim 1 or 2, wherein the selection medium contains: - arsenite: 50 to 500 mg.l-1, - ammonium: = 14 mg.l-1, - mineral salts suitable for providing a nutriment function and/or buffering effect of the said medium. 4.- A process as claimed in any of the preceding claims, wherein the selection medium comprises the following compounds : - phosphates of di- and mono- potassium, which also serve as a pH buffer comprised between 0.05 and 2 g.l-1 - non-biologically oxidizable magnesium salt, such as magnesium sulphate between 0.05 and 1 g.l-1 - non-biologically oxidizable calcium salt, such as calcium chloride between 0.05 and 1 g.l-1 - sodium and chloride, notably in the form of sodium chloride or another non-biologically oxidizable salt between 0.05 and 1 g. 1-1 - metals (trace elements): traces 5.-A process for treating media containing arsenic to oxidize arsenites into arsenates, comprising the step of inoculation of these media in a bioreactor, with a bacterial population essentially constituted of bacteria selected by a process as claimed in any of the preceding claims. 6.- A process as claimed in claim 5, wherein the bacteria are inoculated at a ratio of 5xl05 to 5x106 bacteria per ml of final medium, preferably of the order of 106 bacteria/ml. 7.- A process as claimed in claim 5 or 6, wherein the bacteria are at least partly concentrated in the bioreactor by a support on which the bacteria may adhere. 8.- A process as claimed in any of claims 5 to 7, wherein, to accelerate the oxidation, mineral salts serving as nutritive elements are added to the aqueous medium to be treated and/or air is injected. 9.- A process as claimed in claim 8, in which the following mineral salts are added : - ammonium = 14 mg.l-1, i.e. 10 mg.l-1 of nitrogen; - phosphate of di- or mono- potassium, which also serve as a pH buffer comprised between 0.05 and 2 g.l-1 - non-biologically oxidizable magnesium salt, such as sulphate of magnesium between 0.05 and 1 g.1-1 - non-biologically oxidizable calcium salt, such as calcium chloride between 0.05 and 1 g.l-1 - sodium and chloride, notably in the form of sodium chloride or another non-biologically oxidizable salt between 0.05 and 1 g.l-1 - metals (trace elements) traces. 10.-A process as claimed in any of claims 5 to 9, wherein the microbiological treatment is effected in batch mode or continuously. 11.- A process as claimed in any of claims 5 to 10, wherein the treated media is chosen from : water intended for human consumption, public supply or private drillings, as well as mining or industrial effluents, or underground water. There is disclosed a process for selecting bacteria suitable for oxidizing arsenic in the form of As(III) into arsenate As(V) using CO2 as the sole source of carbon and the As (III) as the sole source of energy, said bacteria presenting in addition at least one of the following characteristics: - they withstand up to at least 1 g.l-1 of As (III) without losing their ability for oxidizing the As (III), the oxidation rate of the As (III) by these bacteria, in a bioreactor, reaches at least 4 mg.1-1.h-1, As (III) concentration being comprised between 100 and 1000 mg.l-1 and bacteria concentration being 106 bacteria per ml, their specific activity of oxidising the As(III) reaches at least 5x10-7 mg of As (III) per bacterium per hour. - they are capable of reducing the residual concentration of As(III) to less than 50 mg.1-1, said process comprising the steps of: b) placement of a sample capable of containing such bacteria in contact with a selection medium containing from 50 to 500 mg. 1-1 of arsenite As(III) and mineral salts; b) placement of the sample and the selection medium in a culture in sterile conditions, in pH conditions comprised between 5.5 and 6.5 and of conditions of temperature comprised between 20°C and 35°C; c) selection of suitable bacteria for oxidizing the As(III) into As(V), if necessary by successive sub-samplings in the selection medium.

Documents:

00469-kolnp-2003-abstract.pdf

00469-kolnp-2003-assignment.pdf

00469-kolnp-2003-claims.pdf

00469-kolnp-2003-correspondence.pdf

00469-kolnp-2003-description (complete).pdf

00469-kolnp-2003-drawings.pdf

00469-kolnp-2003-form 1.pdf

00469-kolnp-2003-form 13.pdf

00469-kolnp-2003-form 18.pdf

00469-kolnp-2003-form 3.pdf

00469-kolnp-2003-form 5.pdf

00469-kolnp-2003-gpa.pdf

00469-kolnp-2003-letter patent.pdf

00469-kolnp-2003-reply first examination report.pdf

469-KOLNP-2003-(05-01-2012)-FORM-27.pdf

469-KOLNP-2003-CORRESPONDENCE 1.1.pdf

469-KOLNP-2003-FORM 27.pdf