Strains of bacteria use of hplc technique b 378, use of hplc technique b 406 and use of hplc technique b 407, reducing sulfate ions to sulfide ions, and the way bacterial decontamination sulfate and metal-containing water

 

(57) Abstract:

The invention relates to new strains of gram-negative bacteria use of HPLC technique In 378, the use of HPLC technique In 406 and use of HPLC technique In 407, sulfate reducing, growing in anaerobic conditions, and method of bacterial disinfection of wastewater, restoring sulfate to sulfide. The strains used methanol as a source of carbon and energy and convert sulfates to sulfides, without the need for other sources of carbon and energy. The method includes the use of the above strains for the recovery of sulphate to sulphide, 3 - 45°C, pH of 3.9 to 9.5 in anaerobic conditions. The method can be used to decontaminate the anaerobic, sulfate, metal-containing and contaminated with radioactive substances in water, in particular for decontamination of mine water and water flooding closed uranium mines, as well as flushing water plants to clean soil, and the process is extremely economical and safe from the point of view of environmental protection in ecologically unfavorable regions. 4 C. and 3 h.p. f-crystals, 3 ill.

The invention concerns a microbiological decontamination sulfate and anaerobic waters and waters contaminated with metals and possibly radioactive substances, with PNA can be used for rehabilitation of mine waters and waters, filling abandoned mines, particularly uranium mines.

The recovery of sulphate waters and waters contaminated with metals, can be done in different ways. The most developed are still methods based on physical and chemical principles. For example, for cleaning sulfate waters perhaps the use of ion-anyoneeven, but this method is not suitable for water treatment, filling abandoned mines, due to the need for processing large amounts of water. In addition, the neutralization and precipitation. A disadvantage is the large instability of operation as a result of their biogarantie [Holl, W. and Kiehling, C. (1979) Nitrat - und Sulfatentfernung aus Rohwassern durch Anionenaustausch, Vom Wasser 53, pp. 189-202; Brettschneider, U. (1990), Die Bedeutung von Sulfaten in der Siedlungswasserwirt-all und ihre Entfemung durch Desulfurikanten, dissertation, Darmstadt).

The method of reverse osmosis pre-treatment and conditioning of purified water. There is a problem of clogging of the membranes, the method is not suitable for processing large quantities of water [Bergmann, F. (1984) Umkehrosmose zur Sulfatentfernung, Wasser, 105. page 217-240; Bergman, F., Ruffer, H., Schneegans, R. and Slomka, T. (1985), Erste first experiences mit der Urnkehrosmose-Anlage Duderstadt zur Sulfatentfernung, Vom Wasser 64, pages 155-167].

Described still by etessami, require a large amount of expensive hardware that isn't working at acidic hydrogen indices and using as carbon source lactate, acetate, or ethanol [Cork, D. C. and Cusanovich, M. A. (1978), Sulfate decomposition, a microbiological process. Waste Treatment and Environmental Considerations, pages 207-221; Cork, D. C. and Cusanovich, M. A. (1979). Continuos disposal of sulfate by a bacterial mutualism. Rev. Ind. Environ, 20, pp. 591-602; Spisac, J. F. (1979), Mettallurgical effluents-growing challenges for second generation treatment, Rev. Ind. Environ. 20. pages 379-387; Uphaus, R. A., Grimm and D. Core, D. J. (1983), Gupsum bioconversion to sulphur: a two-step microbiological process, Rev. Ind. Environ. 24, pages 435-442; Maree, J. P. , Gerber, A., McLaren, A. R., and Hill, E. (1987), Biological treatment of mining effluents, Environ. Technol. Lett. 8, pp. 53-64; Maree, J. P., and Hill, E. (1989). Biological removal of sulphate from indastrial effluents and concomitant production of sulphur, Water Sci. Technol. 21. pages 265-276; Tommerdich, D (1993), Entwicklung a biotechnologischen Verfahrens zur Behandlung saurer sulfat - und metallhaltiger Wasser, PhD thesis, Bonn].

Another method, described in DE 41 06 781 AND 1 is in the making in the dumps sludge wastewater treatment plants as nutrients for the sulfate reducers. This method, however, has several disadvantages. In connection with the introduction of nutrients in the dumps process is uncontrolled and depends on the infiltration of nutrients. For the extraction of metals and recovery of sulphate from mine waters Yes the objective and budget decontamination much sulfuric acid (hydrogen indices from 1 to 2) and metal-containing water-based one-stage process in fermentor. Strains of bacteria, in particular, should be well able to adsorb and accumulate metals, including radioactive elements such as uranium and radium, in order that they could be used also for treatment of mine waters closed uranium mines.

Found new mesophilic gram-negative strains of bacteria growing under anaerobic conditions at a pH of 3.9 to 9.5, mostly from 4 to 9, and at 3 - 45oC, reducing sulfate ions in the sulfite ions. In particular, strains of bacteria, according to the invention, able to use methanol as a source of carbon and energy and do not need additional sources of carbon and energy. In addition to methanol, these strains of bacteria can use for their livelihoods and donors electrodes, such as pyruvate, lactate, acetate, ethanol, butanol, propanol, choline, betaine, succinate, fumarate and benzoate without added hydrogen.

The bacterial strains according to the invention are resistant to metals, they adsorb and accumulate along with heavy metals, such as iron, and radioactive metals, such as, for example, uranium and radium, and light metals such as aluminum.

It is known that in the PEM recovery of water-soluble sulfates in H2S and/or water-insoluble sulfides engaged in energy metabolism in anaerobic conditions and prefer to metabolism of low molecular weight organic substances (CHO) as electron donor.

There are in the literature and some publications about bacteria, reducing sulfate ions, which are able to use methanol as an electron donor. However, none described in the literature strains are not identical with strains according to the invention, and need not only methanol as a source of carbon and energy without additional consumption of carbon and energy from other sources or vitamins.

So, Zellner et al., Arch. Environ, 152 (1989), pp. 329-334, write that kind of marine bacteria Desulfovibrio salexigens is growing on methanol, but this needs to be sodium chloride. Desulfotomaculum orientis is also a form of learning methanol [Klemps et al., Arch. Environ. 143. (1985), pages 203-208] . In contrast to the strains according to the invention, in this case, however, gram-positive strains, unable to assimilate acetate. Isaksen et al., describe in FEMS Microb. Ecol. 14(1994), pages 1-8, strain P60, which, however, is thermophilic, growing optimally at temperatures in the 63o

The strains according to the invention were isolated from sludge of a sewage pond closed factories for the processing of sugar beets in Hermsdorf, near Halle/Germany. The selection was carried out according to conventional and known in the art methods, the cultivation of the strains was generally accepted in the Microbiology of anaerobic methods.

The strains according to the invention, are characterized by a wide pH range of 3.9 to 9 and the temperature range 3 - 45oC, mostly 5 - 40oC, at which they grow and restore the sulfate ions to sulfide ions. The maximum increase is from 0.2 to 0.3 h-. The optimum temperature is 25-30oC, the optimum pH is about 7.

The growth rate was determined on the basis of protein samples in Bradford/Bradford/ (Anal.Biochem. 72, pp. 248-254, 1976 korostil growth at acidic pH below 4,9 or alkaline indicators over 7.5.

As preferred were isolated three strains are indicated by use of HPLC technique In 378, the use of HPLC technique In 406 and use of HPLC technique In 407, which was registered on June 13, 1995 (13.06.95) at DSM (Deutsche Sammlung von Bakterien und Zellkulturen GmbH, Braunschweig/Germany/ German collection of bacteria and cell cultures GmbH, Braunschweig/Germany/) for NN 10041 (use of HPLC technique In 378), 10042 (use of HPLC technique 406) and 10043 (use of HPLC technique In 407).

As already described, the bacterial strains according to the invention, are anaerobic methylotrophic reducing sulfates.

Cells of strain use of HPLC technique In 378 - straight or curved rods, motile. Individual bacteria have a length of 1.5 - 3.5 µm and a width of 0.5 μm. Growth occurs at pH 4 - 9 3 - 40oC. the Maximum growth rate is about 0,25 h-1. The content of G + C in DNA, established using HPLC/liquid chromatography high pressure according to the method of Mesbah et al.Int. J. System. Bacteriol. 39, (1989), pages 159-167, is 58.7 mol %.

Cells of strain use of HPLC technique In 406 - vibrios length of 2.9 - 3,9 µm and a width of 1.1 to 1.5 μm. Growth occurs at pH 6 - 8. The maximum growth rate is about 0,24 h-1.

Cells of strain use of HPLC technique In 407 also have visioneering the form of individual bacteria have a length of 3.1 to 4.0 μm and a width of 1.2 to 1.5 μm. Reproduction occurs at pH 6 to 9, the maximum growth rate zastaivaetsya as a source of carbon and energy, and sulfate as electron acceptor.

Bacteria, according to the invention have excellent suitability for decontamination anaerobic, sulfate, metallic and may be contaminated by radioactive substances water. For example, mine water, filling the closed uranium mines, have high radioactivity due to the presence of natural uranium and radium, their pH is 1 to 2, the water has a high content of sulphate and metals such as iron and aluminum. The mines when they are closed are filled with water, leaving the surface water must be disinfected before it enters waterways.

Therefore, according to the invention, proposes a method of recovery using found bacteria in this water sulfate in H2S (in anaerobic conditions in fermentor), resulting in increased pH. The recovery of sulphate ions and the increase of pH is shown in Fig. 1 and 2 in the example of the use of HPLC technique strain In 378. As a result of increasing pH heavy metal ions are precipitated as insoluble sulfides. Sulfate in this process plays the role of final electron acceptor. The resulting biomass lespedeza efficiency of the process is important, the bacteria had the ability to use inexpensive source of carbon and energy, which is available in any quantity. This source is methanol. Its use is a particular advantage of this process. If methanol is used only for the recovery of sulphate, in accordance with the following chemometric:

4 CH3HE IS + 3 SO42-+ 2H+---> 4HCO3+3 H2S + 4N2O

would of 0.44 g of methanol, 1 g of sulfate. Indeed consumed more methanol (see Fig. 3). By using the energy obtained on the basis of the specified redox reaction for biosynthesis occurs assimilation (carbon) of methanol, which acts as a bacterial biomass, the formation of which is desirable due to the fact that it is, on the one hand, is needed as a catalyst, and on the other hand, acts as a "drive or adsorber heavy metals.

Due to the fact that energy is produced in small quantities, requires a significant amount of methanol oxidation for the synthesis of one unit of biomass, i.e. for the implementation of the growth and reproduction of a population of bacteria. Depending on the energy consumption of methanol for vos the tat as a source of carbon and energy, what is conceptually possible, according to theoretical estimates, for recovery 1 g of sulfate would be required not less 0,73 g

The same amount would be required in the case of lactate, source of carbon and energy for many species of bacteria, sulfate reducing.

The comparison confirms the advantage of using methanol. Considering the price of methanol, on the one hand, and on the acetate or lactate, on the other hand, the advantage of using methanol becomes even more obvious.

The invention therefore also concerns the method for bacterial decontamination sulfate and metal-containing water by the use of strains of bacteria, reducing sulfate ions.

Decontamination sulfate and metal-containing water can be carried out in batch modes and ferbatch, as well as in continuous mode. The process of decontamination can occur at temperatures of from 3 to 40oC, mostly between 25 and 30oC. In accordance with the invention, for disinfection of sulfate-and metal-containing mine water and flooding the mine waters are suitable not only new strains of bacteria as such, but also their mixtures.

The horse is received as a wash for plants to clean soil, if the soil decontamination is treated with sulfuric acid.

The industrial method of treatment of polluted water is beneficial to implement in the reactor, for example in the boiler with a mixer that allows you to control and manage them, i.e. to optimize the process parameters, such as temperature, and the supply source of carbon and energy in order to create the best conditions for the recovery of sulphate and for the growth of beneficial bacteria strains.

Fig 1 - 3 indicate that:

Fig. 1 - recovery of sulphate in the culture of strain use of HPLC technique In 378, in batch mode (the determination on the basis of three cultures);

Fig. 2 is a representative example of increasing pH over time in cultures of strain use of HPLC technique In 378, in batch mode is:

the initial pH of 6.5, 10 mm methanol;

the initial pH of 4.5; 10 mm methanol;

the initial pH of 4.3; 10 mm methanol;

the initial pH of 5.0; 31,25 mm methanol;

Fig. 3 - growth curve of a culture of strain use of HPLC technique In 378 in batch mode with 10 mm methanol as a source of carbon and energy, determination on the basis of three cultures:

protein;

the methanol.

Below the invention is described by examples that it is is in the fermenter under anaerobic conditions with a flow of nitrogen at 30oC and at a pH of 7.

Nutrient solution is composed of three solutions:

Solution 1: 0.5 g FeSO47H2in 10 ml of distilled H2O;

Solution 2: 0.1 g of thioglycolic, 0.1 g of ascorbic acid, 20 mg of dithionite in 10 ml of distilled H2O;

Solution 3: 10 ml of 1 M methanol, 2.0 g MgSO47H2O,

1.0 g CaSO4, 1.0 g NH4Cl, 0,5 KH2PO4,

1.0 g yeast extract in 1 l of distilled H2O.

You can cultivate the strain and without the addition of yeast extract.

During breeding this strain of bacteria on methanol as carbon source and energy recovering sulfate with the formation of sulfide. It consumes about 9.5 g of methanol and 1 g is formed biomass. The resulting ratio 0.66 methanol: 1 SO4(g/g) is very close to the estimated ones of 0.54:1.

When using, according to the invention, the bacterial strain for decontamination sulfate and metal-containing water use recovery solution 2 is required only if the waters are not anaerobic, i.e., if no reducing conditions. Components of solution 3, such as phosphates and chlorides, as pravila as a source of carbon and energy. You can add yeast to accelerate growth.

Example 2

Bacterial strain use of HPLC technique In 378 analogously to example 1 are cultivated on methanol as carbon source and energy, and in contrast to example 1 each pH is set at 6.3. Under these conditions, the difference in speed specific consumption of sulfate in the ratio of methanol:sulfate is not observed. However, it is expected stoichiometry of neutralization, the pH within three weeks increases to 7,05, and after a further 7 days to reach even 8,0.

Example 3

Bacterial strain use of HPLC technique In 378 cultivated as described in example 1. In this case, the original pH is 4.3. Under these conditions, the deterioration in the ratio of methanol to sulfate. Efficiency growth unexpectedly increases, the pH increases rapidly after one week, he comes to 6, after 4 weeks to 6.6.

Example 4

Bacterial strain use of HPLC technique In 378 analogously to example 1 are cultivated on methanol as carbon source and energy. The specific rate of recovery of sulfate is about 0.7 g/g/H. this implies that the rate of formation of sulfide in 0,23 g/g/h In the corresponding flooding waters of the iron content is approximately 500 mg/L. In this example, is,82 g of iron in the form of a sulfide precipitate, and after 24 h even 9,84 g Fe per 1 g of biomass.

Example 5

Bacterial strain use of HPLC technique In 378 propagated as in example 1. In contrast to this example in this case is added to the Al2(SO4)3at 10 mm. The concentration of dissolved aluminum is 1.05 mg/l, the recovery of sulfate occurs at a rate of approximately 0.8 g/g/H. With this aluminum is released from the solution by adsorption and accumulation. By analysis it was found that the biomass of the loaded number of 0.69 mg/l, of which the quantity of 0.62 mg/l is placed on the cell membrane.

The composition of the metal-containing water in example 5, the following (concentration - mg/l): Al - 1,05; Ca - 398,2; Cd - 0,5; CE - 0,62; -4,42; Cr - 0,2; Cs - 0,03; Cu - 40,63; F - 2910,0; - to 5.21; Li-53; Mg - 1416,0; Mn - 137,9; Na - 24,0; Ni - or 115.1; P - 1,81; Pb - 0,51; Ti - 0,3; U - 4,3; V - 4,9; Zn - 45,08; Sulfat - 20283,0; Chlorid - 11,4.

Example 6

Strain the use of HPLC technique In 378 grows as described in example 1. The composition of the metal-containing water, which must be decontaminated, given above (example 5). The concentration of dissolved aluminum - 1,05 mg/l Sulfate is reduced in the range of 1.6 g/g/ hour, aluminum is removed from solution by adsorption and accumulation. The analysis showed that the biomass loaded amount of 0.52 mg/l, of which the number of 0.45 mg/l posted on cellular obatala should be disinfected, given above (example 5). The concentration of dissolved aluminum - 1,05 mg/l Sulfate is reduced in the range of 1.7 g/g/h, the aluminum is removed from solution by adsorption and accumulation. The analysis showed that the biomass loaded number 0,63 mg/l, of which the number of 0.59 mg/l is placed on the cell membrane.

In the following examples 8, 9 and 10 show the disinfection cleansing water treatment plants, soil following composition (concentration - mg/l): Al - 1090,5; - 442,36; Fe - 860,0; - 16,9; Mg -4832,47; Mn - 0,9; Na - 54,0; U - 1,6; Sulfat - 19252,05; Chlorid - 57,1.

Example 8

Strain the use of HPLC technique In 378 grows, as described in example 1, in methanol as the sole source of carbon and energy. Sulfate is reduced in the range of about 2.4 g/g/h, the concentration of magnesium decreases from 4832 to 2792,16 mg/l, the concentration of manganese is reduced from 0.9 to 0.07 mg/l and the concentration of iron from 860 to 12.9 mg/l and uranium removed 98%.

Example 9

Strain the use of HPLC technique In 406 grows, as described in example 1, in methanol as the sole source of carbon and energy. Sulfate is reduced in the range of about 1.7 g/g/h, the concentration of magnesium decreases from 4832 to 3486,16 mg/l, the concentration of manganese is reduced from 0.9 to 0.12 mg/l and the concentration of iron from 860 to 64.5 mg/l and uranium removed 98%.

Prima and energy. Sulfate is reduced in the range of about 2.25 g/g/h, the concentration of magnesium decreases from 4832 to 2202,32 mg/l, the concentration of manganese decreases from 0.9 to 0.1 mg/l and the concentration of iron from 860 to 36.4 mg/l and uranium removed 98%.

In example 4, and in the following examples 11 and 12 were obezzarajivatei flooding closed uranium mines water of the following composition (concentration - mg/l): Al - 768,5; Ca - 200,0; Fe - 500,0; It Is 1.5; Mg - 1854,0; Mn - 179,0; Na - 17,0; U - 8,5; Sulfat - 14792,0; Chlorid - 10,6.

Example 11

Strain the use of HPLC technique In 378 grows, as described in example 1, in methanol as the sole source of carbon and energy. Sulfate is reduced in the range of about 2.8 g/g/h, the concentration of magnesium is reduced by 63%, manganese - 92.5%, iron - on 98% and uranium - 99% (same as in example 4).

Example 12

Strain the use of HPLC technique In 407 grows, as described in example 1, in methanol as the sole source of carbon and energy. Sulfate is reduced within about 2,75 g/g/h, the concentration of magnesium is reduced by 61%, manganese - 92%, iron - on 97% and uranium - 99%.

1. Bacterial strain use of HPLC technique In 378, deposited under DSM 10041, reducing the sulfate ions to sulfide ions.

2. Bacterial strain use of HPLC technique In 406 deposited under DSM 10042, reducing the sulfate ions to sulfide ions.

4. Method bacterial decontamination sulfate and metal-containing water using sulfate-reducing bacteria, characterized in that use strain of bacteria on PP.1, 2 or 3, the process is carried out in anaerobic conditions at a pH of 3.9 to 9.5 and 3 - 45°C, and along with the conventional nutrient salts strain of bacteria, reducing sulfate ions, add only methanol as the sole source of carbon and energy.

5. The method according to p. 4, characterized in that use strain that is able to adsorb and accumulate radioactive, heavy and light metals.

6. The method according to p. 4, characterized in that the neutralization is subjected to a wash water generated at the treatment plants of the soil.

7. The method according to p. 4, characterized in that the neutralization is subjected to much sulfuric acid and metal-containing water and water contaminated with radioactive substances.

 

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