Classifications

• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/02—Aerobic processes
  • C02F3/10—Packings; Fillings; Grids
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/30—Aerobic and anaerobic processes
  • C02F3/302—Nitrification and denitrification treatment
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W10/00—Technologies for wastewater treatment
  • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

• the invention relates to a method for nitrification and nitrogen elimination by means of nitrifying bacteria.
• nitrification is understood as the bioi ⁇ gi- cal conversion of ammonia via nitrite to nitrate.
• two groups of bacteria are responsible for this, which are summarized under the generic term nitrificants.
• ammonia oxidants oxidize ammonium to nitrite
• nitrite oxidants oxidize nitrite to nitrate.
• nitrificants are of great industrial importance.
• Your metabolic rate is used e.g. B. in sewage treatment plants, in which they ensure the implementation of ammonia in individual nitrification stages.
• the disadvantage is that after nitrification has taken place, the actual nitrogen elimination has not yet been achieved. Rather, the nitrogen is in the form of nitrate. Using a separate and technically more complex process, denitrification, nitrate is then converted with the release of nitrogen. However, this process is very cost-intensive and is therefore only used in a few sewage treatment plants.
• the object of the invention is to improve the above-mentioned method so that nitrification and nitrogen elimination is possible in a simple and inexpensive manner by means of nitrifying bacteria.
• nitrificants it should be possible, starting from an aqueous suspension of nitrificants, to bind the bacteria in a metabolically physiologically active state to a carrier material, to dry the bacteria in a storable manner, so that, if necessary, the carrier material together with the bacteria is added to it from an aqueous solution exposing eliminating ammonium, nitrite, and / or nitrate, the bacteria inactivated for a short or longer period by water removal on the carrier material reactivating their nitrification-specific enzyme system and resume their nitrification performance as free immortalized or reproducing cells that are detached from the carrier material.
• the object of the invention is to cultivate such a strain of nitrifying bacteria which is suitable on the one hand for producing high cell yields and on the other hand can subsequently be converted to mineral or medium contaminated with dissolved organic substances when grown under these conditions, without resulting in a long lag phase in order to resume the nitrification performance or without reactivating the bacteria at all.
• the object is achieved by starting from a nitrate-containing aqueous solution by reducing the nitrate by nitrite oxidants, oxidizing the reaction products ammonium by ammonia oxidants and nitrite by nitrite oxidants in a continuous process to nitrate and thereby gaseous nitrogen compounds such as N2O or NO are released, as well as through the selection and use of the microorganism Nitrobacter nov. spec. T3.
• nitrate is reduced by nitrite oxidants, in particular of the genus Nitrobacter. If these organisms are used exclusively, the small amounts of ammonium produced will not be oxidized again. For optimal nitrogen elimination, it is therefore advantageous to use not only nitrite oxidants but also ammonia oxidants, for example of the genera Nitroso onas r Nitrosovibrio or Nitrosospira. The ammonium which is noticeable or already present in the nitrate or nitrite reduction is then included in the reaction cycle.
• the optimal nutrient medium for the strain in question is selected for growing the nitrificants and for producing high cell yields. The following media are available for this, but their composition can still be changed.
• NaCl can be added to this medium in a concentration of 10-200 mM.
• Lithoautotrophic media are used exclusively for the growth of ammonia oxidants, since heterotrophic growing ammonia oxidants have so far not been isolated.
• the media for growing ammonia oxidants can differ significantly from one another in terms of their salinity.
• the differentiation within this organism group between land, sea and brackish water strains is common. Accordingly, the respective synthetic medium is adapted to the natural conditions.
• nitrite oxidants In contrast to ammonia oxidants, nitrite oxidants also grow in the presence of organic substrates. With mixotrophic or heterotrophic growth, some species even deliver significantly higher cell yields than under purely chemolithoautotrophic conditions. For example, the species Nitrobacter hamburgensis is characterized by this property (Arch. Microbiol. 136, 281-283). An ⁇ under mixotrophic growth conditions of the cell yield values between 'reaches 30 and 35 mg total cell protein / first The cell yield is therefore more than ten times higher than under purely chemolithoautotrophic conditions.
• Nitrite oxidants that show their optimum growth under heterotrophic conditions are not yet known and will be further described for the first time by the new species Nitrobacter nov. spec. T3 described.
• Nitrificants are therefore cultivated under optimal growth conditions and then concentrated to increase the cell density. The concentrate is then resuspended. Basically, are suitable for Resuspension so-called "washing solutions", which in their
• compositions except for the substance representing the energy source correspond to the respective nutrient media.
• the bacteria are preferably adjusted to a cell concentration of 10 11 to 10 12 cells / ml. Such suspensions can then be used to apply carrier materials or used directly for the process.
• Open-pore porous materials are suitable as carrier materials for immobilizing the nitrifi edges. These include activated carbon with an internal surface area between 500 and 1500 m 2 / g, which is preferably used in granular form, and zeolites, which belong to the silicate minerals.
• ion exchangers consisting of organic solids are also suitable as carrier materials. They generally have a hydrophilic gel structure with a large surface area and are particularly suitable as macroporous ion exchangers. Ceramics can also be used as the carrier material.
• the carrier materials are incubated in the resuspension solutions with the cell concentrations of 10 11 to 10 12 cells / ml. Incubation takes place at a temperature in the range of 20-30 ° C. 1-5 ml of the bacterial suspension are added to carrier materials with a volume of 10 cm 3 until all of the liquid has been absorbed by the carrier material. If the absorption capacity is large, the amount of bacterial suspension used can be increased until the maximum absorption capacity of the carrier material is reached.
• the carrier loaded with bacterial suspension is then dried arerially.
• the material is stored for a period of, for example, 5 days at a temperature between 18 and 25 ° C., so that a gentle and uniform release of water takes place, the drying can also be accelerated by the fact that the loaded carrier material is under vacuum in corresponding vacuum vessels is dried. As a result, the drying process can be shortened to 24 hours.
• the dried and bacterially loaded carrier material is to be kept in a dry place for further storage, if possible. Storage in evacuated and welded plastic bags is recommended, which can then be stored at room temperature or at 4 ° C.
• the loaded carrier material can be used directly if required. It should be added to the aqueous solution at the optimal growth temperature in which the biological conversion processes are to take place. At a suboptimal temperature, the nitrification process slows down.
• active precultures can also be generated using the dried material by inoculating fresh synthetic nutrient medium first and then adding this culture to the aqueous solution to be cleaned. In both cases, it is not necessary for the bacteria to remain immobilized on the carrier in order to absorb their nitrification performance. They can also detach and become metabolically physiologically active as freely moving cells.
• the process according to the invention can be used to combine the previously multistage processes in wastewater purification and drinking water treatment which, in addition to a nitrification stage, have a denitrification stage which is spatially separate therefrom.
• the total outlay for nitrogen removal can thereby be reduced considerably.
• nitrifying agents can be induced to reduce nitrate or nitrite and to release gaseous nitrogen compounds such as N 2 O or NO.
• a decisive factor here is a reduction in the oxygen partial pressure in the culture medium. Both processes, the nitrate or nitrite reduction and the nitrification run side by side, whereby zones of different, ie higher and lower, oxygen partial pressures result in cultures that are not or only slightly stirred. The nitrification then takes place in the zones of higher oxygen partial pressures, while the nitrite or nitrate reduction takes place in zones of lower oxygen partial pressures.
• the nitrificants immobilized according to the invention can also be used for such a method for coupled nitrification / nitrite or nitrate reduction.
• the particular advantage of immobilized cells results from the fact that the microorganisms can be directed to the locations of certain oxygen partial pressures and brought there to the corresponding metabolic rate. Nitrogen elimination can thus * be achieved in one step with relatively simple means without changing the milieu conditions.
• the process for nitrification and nitrogen elimination is not only suitable for wastewater treatment and
• Drinking water treatment can be used in practically all bodies of water in which the nitrogen pollution is too high. It is essential that in ammonium, A combined nitrification / nitrogen elimination is carried out in situ by adding the bacterial material to nitrite and / or nitrate contaminated natural or artificial waters.
• the area of aquaculture is becoming increasingly interesting. Intensive fish farming regularly leads to toxic nitrite concentrations that cause fish to die. Using the nitrifying agents, the nitrite can now be broken down and nitrogen can be removed from the water at the same time.
• all nitrifying agents are suitable for the method according to the invention.
• relatively high cell concentrations have to be used for effective use. These, in turn, can only be produced from litho-autotrophic cultivations with considerable and economically unjustifiable effort. It is therefore advisable to cultivate nitrificants mixotrophically or heterotrophically.
• the resulting cell yields which, in contrast to lithoautotrophic cultivation, are more than ten times higher, are sufficient to apply the carrier material.
• Nitrobacter nov. spec. T3 behaves completely different. This organism was selected on mineral medium over a period of one year. It hardly differs from morphologically previous species, but shows clear physiological, biochemical and genetic peculiarities. Its main distinguishing feature is the growth behavior on a heterotrophic medium and in the presence of nitrite. Special features of this organism are
• the plasmid DNA is in vivo in a covalently closed circular form and, based on the electrophoretic migration behavior, has a 0.5% strength
• Agarose gel the molecular weight 80 + 3 MD. It can be obtained by in vivo replication in the microorganism of the genus Nitrobacter described above by aerobic or micro-aerophilic cultivation of the cells under lithoautotrophic, ixotrophic or heterotrophic culture conditions. The plasmids can be replicated identically without additional selection pressure and transferred to daughter cells.
• 5 shows the schematic representation of an electrophoretic separation of the membrane proteins from N. nov. spec. T3 compared to N ⁇ hamburgensis X14 and
• FIG. 6 shows the schematic representation of a gel-electrophoretic separation of the DNA from ⁇ nov. spec. T3 compared to Nitrobacter hamburgensis X14.
• Fig. 1 the growth curve of Nitrobacter nov. spec. T3 shown after heterotrophic cultivation. This curve shows a typical lag phase at the beginning of the course, a subsequent logarithmic phase and finally a stationary phase with a plateau after 45 days.
• the cell yield achieved with 50 mg total cell protein / 1 is about 60% higher than with N. hamburgensis, which showed the best growth of all strains of nitrifying bacteria.
• FIG. 2 shows Another physiological peculiarity of the new species N. nov. spec. T3 in Figures 2 to 4. They show growth curves for this organism in the presence of different nitrite concentrations. In all cases, a decrease in the nitrite concentration is accompanied by an increase in protein, with the cell yield achieved after 45 days being lower compared to heterotrophic cultivation with increasing initial concentration of nitrite.
• Fig. 2 shows the growth at an initial nitrite concentration of 0.5 g NaN0 2 / l. After 45 days, the cell yield reaches a comparative value of approximately 36 mg total cell protein / 1 and increases further to approximately 38 mg total cell protein / 1. The nitrite used is oxidized after 15 days.
• the nitrite concentration used significantly inhibits growth.
• the nitrite used is only used up after 29 days.
• An extreme inhibition of growth is illustrated in FIG. 4.
• the nitrite used is oxidized with approximately the same conversion rate as in the experiments shown in FIGS. 2 and 3, the protein content hardly increases here. Only after 39 days when the nitrite concentration had dropped to 0.5 g NaN0 2 / l was it possible to measure a slight increase in protein.
• the comparison value after 45 days is approximately 13 mg of total cell protein / 1 and is therefore a factor of 4 lower than that of heterotrophic cultivation.
• This growth behavior underlines the strong heterotrophic potency of this type, which, however, does not come at the expense of the lithoautotrophic growth.
• This organism grows chemolithoautotrophically in comparison to other representatives of this genus.
• N. nov. spec. T3 characterized by the fact that only relatively short adaptation times are required in order to nitrify in the case of changed oxygen partial pressures, in particular increases.
• the disadvantage of a lack of adaptation performance in conventional species affects, as already shown, the method according to the invention. By using this new bacterium, the impairment due to "oxygen stress" in nitrite oxidants is overcome for the first time.
• Nitrite oxidoreductase is the membrane-bound key enzyme of nitrite oxidation. According to previous knowledge, it can be induced both by nitrite and by nitrate. However, its activity can be
• N. nov. spec. T3 is also a biochemical peculiarity, which is evident in a gel electrophoretic separation of the membrane proteins.
• Figure 5 shows SDS polyacrylamide gel electrophoresis of these proteins.
• the large subunit of nitrite oxidoreductase has a relative molecular weight of 115,000 in all strains examined to date.
• the molecular weight deviates from this and is approximately 130,000.
• other membrane proteins are also changed in their relative molecular weight.
• the second subunit of the terminal oxidase, the cytochrome aa 3 has in N. nov. spec.
• T3 has a relative molecular weight of around 27,000, which is about 1,000 less than in the other cases.
• the membrane-bound cytochrome c after gel electrophoretic separation is somewhat above the comparison band from N ⁇ hamburgensis X14, which is also typical for all other species.
• N. nov. spec. T3 further characterized by having a large plasmid.
• Nitrobacter plasmids have so far only been detected in strains of the type N. hamburgen ⁇ sis.
• all the strains of the species N ⁇ winogradskyi examined are plasmid-free. Over time, this fact has led to the interpretation of the possession of at least certain plasmids, for example in the case of N. hamburgensis, as a species characteristic.
• T3 after cesium chloride-ethidium bromide density gradient centrifugation.
• the molecular weight of 80 + 3 MD can be determined from this.
• N. nov. spec. T3 Owning exactly one plasmid is necessary for N. nov. spec. T3 characteristic. It is striking that this plasmid pPB31 is stable even under different growing conditions. A plasmid loss could not be observed even without additional selection pressure. Even the use of mutagenic agents did not lead to the appearance of plasmid-free mutants. It can therefore be assumed that in the cultivation of N. nov. spec. T3 achieves expression of metabolically physiologically relevant plasmid genes under the specified conditions and thus a selection pressure for targeted plasmid replication arises. That N. nov. spec.
• the plasmid function can be connected with the physiological performance of this bacterium.
• a plasmid-free strain will therefore lack the essential metabolic benefits.
• N. nov. spec. T3 not only offers advantages over previously known nitrite oxidants when using the method according to the invention, but is also characterized by long periods of metabolic physiological activity in resting cultures.
• So z. B. cells cultured microaerophilically on a heterotrophic medium are still active after three months. After this and after a long time, they can be left lithotrophic in an oxygen-rich medium cultivate and begin nitrification there after a few days.
• This special metabolic performance, the above-average growth under heterotrophic conditions and the subsequent lithotrophic nitrification without longer adaptation times characterize this organism as well as its oxygen tolerance after microaerophilic cultivation.

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• 1987
  • 1987-07-21 DE DE19873724027 patent/DE3724027A1/de active Granted
• 1988
  • 1988-07-19 KR KR1019890700488A patent/KR890701480A/ko not_active Withdrawn
  • 1988-07-19 WO PCT/EP1988/000651 patent/WO1989000547A1/de not_active Application Discontinuation
  • 1988-07-19 AU AU21319/88A patent/AU2131988A/en not_active Abandoned
  • 1988-07-19 JP JP88506343A patent/JPH03501081A/ja active Pending
  • 1988-07-19 HU HU884769A patent/HUT52742A/hu unknown
  • 1988-07-19 EP EP88907063A patent/EP0377595A1/de not_active Ceased
• 1990
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