US2023143790

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Page 1

COCA A A A
US 20230143790A1
as) United States

a2) Patent Application Publication (1) Pub. No: US 2023/0143790 Al
KORNILOVA et al. (43) Pub. Date: May 11, 2023

(54) METHOD FOR THE AEROBIC AND 0) Foreign Application Priority Data
ANAEROBIC CULTIVATION OF
MICROORGANISMS, METHOD FOR THE Feb. 29,2020 (DE) 10 2020 001 337.6
PRODUCTION OF A PREPARATION FOR
CLEANING RADIOACTIVE LIQUIDS AND Publication Classification
RADIOACTIVELY CHARGED SURFACES,
METHOD FOR CLEANING RADIOACTIVE

LIQUIDS AND METHOD FOR CLEANING G1) Inch (2006 01)
RADIO-ACTIVELY CHARGED SURFACES 2006.
GUF 9/28 (2006.01)

(71) Applicant: Sylvia SCHREIBER, Dornbirn (AT)

- Abi va 62): USC
(72) Inventors Moscone Rt): Vlada vanvich A, CPC ... G2LF 9/18 (2013.01); G21F 9/28 (2013.01)

VYSOTSKIL, Kiev (UA)
(73) Assignee: Sylvia SCHREIBER, Dombim (AT) (57) ABSTRACT
(21) Appl. No.: 17/907,840
(22) PCT Filed: Feb. 24, 2021

The invention relates to a method for aerobic and anaerobic
cultivation of microorganisms. The invention also relates to
a method for producing a preparation for cleaning radioac-

(86) PCT'No.: PCT/EP2021/054616 tive liquids and radioactively charged surfaces. Likewise,
§ 371 (c)(1), the invention further relates to a method for cleaning radio-
(2) Date: Aug. 29, 2022 active liquids and radioactively charged surfaces.

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Patent Application Publication May 11, 2023 Sheet 1 of 4 US 2023/0143790 Al

Fig. 1

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Patent Application Publication May 11, 2023 Sheet 2 of 4 US 2023/0143790 Al

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Patent Application Publication May 11, 2023 Sheet 3 of 4 US 2023/0143790 Al

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Patent Application Publication May 11, 2023 Sheet 4 of 4 US 2023/0143790 Al

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US 2023/0143790 Al

METHOD FOR THE AEROBIC AND
ANAEROBIC CULTIVATION OF
MICROORGANISMS, METHOD FOR THE
PRODUCTION OF A PREPARATION FOR
CLEANING RADIOACTIVE LIQUIDS AND
RADIOACTIVELY CHARGED SURFACES,
METHOD FOR CLEANING RADIOACTIVE
LIQUIDS AND METHOD FOR CLEANING
RADIO-ACTIVELY CHARGED SURFACES

CROSS-REFERENCE TO RELATED
APPLICATIONS

[0001] This application is a national stage under 35 U.S.C.
§371 of PCT Patent Application PCT/EP2021/054616 filed.
on 24 Feb. 2021, which is pending and which is hereby
incorporated by reference in its entirety for all purposes.
PCT/EP2021/054616 claims priority to German Patent
Application 10 2020 001 337.6 filed on 29 Feb. 2020,
which is hereby incorporated by reference in its entirety
for all purposes.

BACKGROUND OF THE INVENTION
1. Field of the Invention

[0002] The invention is directed to a method for the aero-
bic and anaerobic cultivation of microorganisms, a method
for the production of a preparation for cleaning radioactive
liquids and radioactively charged surfaces, and . a method
for cleaning radioactive liquids and radioactively charged
surfaces.

2. Discussion of the Related Art

[0003] Radioactive liquids may accrue in connection with
various, particularly technical processes. The largest source
of radioactive liquids is to be found in connection with the
operation of nuclear power plants. The enormous amounts
of radioactive liquids accruing as waste in nuclear power
plants are a considerable problem for the operators of the
nuclear power plants on the one hand and for life on Earth
on the other hand. On the one hand, the radioactive liquids
have to be temporarily stored in the area of the nuclear
power plants. A final storage of the radioactive liquids has
to take place so that storage times are reached which con-
siderably exceed the half-life of the involved radioactive
isotopes without risk. When thinking of one of the most
common radioactive isotopes encountered in the operation
of nuclear power plants, namely '37Cs and its half-life of
more than 30 years, it is easy to realise how high the chal-
lenges are. While radioactive solids have a small volume
relative to their radioactivity and generally pose just a low
risk of chemical nature, radioactive liquids have a large
volume so that high storage capacities are required, and
the liquids frequently tend to chemically attack the contain-
ers in which they are accommodated and to leak from them
in the worst case. Then, it takes very little until the radio-
active liquid is distributed in an uncontrolled manner, for
example via the ground water, so that extreme damage is
caused to people, animals, and the environment in general.
[0004] Already, concepts were developed how to remove
the radioactive substances from the liquid to thereby pro-
duce a solid which is relatively easy to store and has pre-
cisely this radioactivity. What is known are chemical pro-

May 11, 2023

cesses on the basis of an ion exchange as well as
microbiological systems using radioactive substances for
their growth within the scope of their development. Biolo-
gical systems are insofar superior to the classical systems as
they can develop a high selectivity with regard to the
absorption of radioactive substances which is not necessa-
rily guaranteed in classical chemical systems.

[0005] Further Background: On Earth, about 400 nuclear
power plants are operated. In the process, a large amount of
dangerous radiotoxic waste is generated. Due to the risks
caused thereby, deactivation methods are considered. A
deactivation of radioactive waste is generally highly compli-
cated, cost intensive, and time consuming. Radioactively
contaminated water must not end up in waste water, con-
taminated ground has to be removed and disposed of. Final
disposal sites have to be set up so that the radioactive sub-
stances do not reach the environment and that the radiation
emitted by them will not reach people and the biosphere.
Prior to the final storage, the radioactive waste should be
brought into a form safe for storage. Highly radioactive
liquid waste is cast in steel moulds or ingot moulds with
molten glass. If these containers become brittle or get
damaged under the exposure to radiation and heat an envir-
onmental contamination is possible. In this case, radioactive
gases may reach the atmosphere. The highest risk arises
when the radioactive substances reach the ground water.
The probability of such a catastrophe depends on the geolo-
gical stability of the final disposal site, when it is located in a
salt mine on the ground water flows surrounding the salt
mine, and on the potential changes of the salt structure by
radiation and heat. The containers containing radioactive
substances should be permanently stored in locations
where they can neither be damaged by water nor by earth-
quakes or other influences. Here, it should be taken into
consideration that the high heat emission produced during
the decay of radioactive substances requires an extremely
efficient cooling of this waste over the course of many dec-
ades. Therefore, the final storage of radioactive substances
is a global problem which is difficult to solve. None of the
countries in which nuclear power is used has found a safe
and permanent solution for the safe treatment of radioactive
waste to date. Nuclear power plants only have temporary
storage facilities for spent fuel. In addition, hundreds of
thousands of tons of active water containing various radio-
active isotopes are present in nuclear power plants. This
water is produced in the operation of boiling water and pres-
surised water reactors as well as reactors of other types both
during the long-term decay (deposition) of the fuel elements
in the water after the expiration of the useful life and in the
use of water as a heat transfer medium flowing through the
reactor core. Only after a cool down time of three years in
spent fuel pools of nuclear power plants, the overall activity
of the fuel elements decreases by about one order of magni-
tude due to the decay of the most short-lived isotopes. 99%
of the remaining activity are attributable to relatively long-
lived isotopes of strontium, zirconium, ruthenium, caesium,
iodine and rare earths. A certain activity is also generated by
the isotopes of noble gases. Of particular importance is the
deactivation of caesium radionuclides the proportion of
which in the overall activity of liquid waste reaches 98%.
In the practice of the deactivation of liquid waste of a low
and medium activity level, cleaning methods involving
organic ion exchangers are known. Thus, highly acidic
cation exchangers and highly alkaline anion exchangers

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US 2023/0143790 Al

are used in cleaning reactor blow-down water. For cleaning
water from a basin reactor, ion exchange filters charged with
cation and anion exchangers are used. Further known are
systems comprising organic cation exchanger resins in Na+
form as well as synthetic zeolithe (type A, erionite, chaba-
site, phillipsite). The drawback of sorption methods for
deactivating solutions of medium activity with the aid of
the organic ion exchanger resins is their low radiation resis-
tance. Consequently, there is damage to the basis, the
exchange capacity decreases, and the exchange kinetics
deteriorate. The other disadvantage of the organic resins is
their low selectivity, particularly with regard to caesium
radionuclides such as !57Cs and '4Cs.

SUMMARY OF THE INVENTION

[0006] The invention is based on the object to provide
appropriate microorganisms in a sufficient number to pro-
duce a preparation by which radioactive liquids and radio-
actively charged surfaces can be reliably, efficiently, and
safely cleaned, i.e. freed from their radioactivity on this
basis.

[0007] This object is solved by the features of the indepen-
dent claims. Advantageous embodiments are indicated in
the dependent claims.

[0008] The invention consists in a method for the aerobic
cultivation of microorganisms comprising the steps of:

[0009] providing an aqueous solution having a pH value
of 5.5 to 9.0 in a container,

[0010] adding a substrate in a first substrate dosage to
the aqueous solution,

[0011] adding further elements to the aqueous solution,

[0012] adding an inoculant including microorganisms
to the aqueous solution,

wherein, by the abovementioned steps, an initial product is
provided, and wherein, subsequently, the following steps are
performed:

[0013] introducing air into the initial product and/or into
intermediate products developing therefrom,

[0014] varying a temperature of the initial product and/
or the intermediate products developing therefrom in a
range from 15 to 35° C.,

wherein, after having added the substrate in the first sub-
strate dosage, the following steps for monitoring the cultiva-
tion are performed:

[0015] taking a reference liquid sample and determining
a first concentration of organic substance in the refer-
ence liquid sample,

[0016] taking another liquid sample and determining
another concentration of organic substance in the
further liquid sample after the expiration of the first
waiting time,

[0017] if the further concentration of organic substance
is smaller than 10 percent of the first concentration of
organic substance, adding substrate in another substrate
dosage,

[0018] repeating the abovementioned steps for monitor-
ing the cultivation until a sufficient amount of biomass
is present in the container.

[0019] The underlying aqueous solution has a pH value of
5.5 to 9.0, this value particularly taking account of the fact
that the microorganisms are “aerobic microorganisms”, i.e.
suitable for an aerobic cultivation. The pH value, like all
parameters discussed in the following, may be varied to
thereby optimise the course of the process, be it with regard

May 11, 2023

to the result of the cultivation, the rate of the cultivation, or
the amount of ultimately obtained microbiological material.
A substrate serving the sustenance of the microorganisms is
added to the aqueous solution. Likewise, other elements are
added which are required for the growth of the microorgan-
isms or stimulate the growth of the organisms. Which ele-
ments this could be will be specified in more detail below.
Important for the initial product of the cultivation is the
addition of an inoculant including microorganisms. These
may originate from various sources, for example from ear-
lier cultivations obtained in an identical or similar way, or
also primarily from other sources, for example from sewage
treatment plants. As soon as the initial product is available it
may serve the cultivation of microorganisms. Since the cul-
tivation takes place aerobically air is introduced. Further-
more, the temperature of the initial product or the intermedi-
ate products developing therefrom is varied, a temperature
range of 15 to 25° C. being instrumental for this purpose.
Often, temperatures in the range of 23 to 25 degrees are
optimal. The initial product or the intermediate products
developing therefrom are then monitored to finally obtain
a sufficient amount of biomass. To this end, samples are
taken, and, depending on the obtained concentrations of
organic substance at the various points in time, further sub-
strate for sustaining the cultures is added. When finally a
sufficient amount of biomass is present, it can be withdrawn
for further processing.

[0020] The invention further relates to a method for the
anaerobic cultivation of microorganisms comprising the
steps of:

[0021] providing an aqueous solution having a pH value
of 4.5 to 7.5 in a container,

[0022] adding a substrate in a first substrate dosage to
the aqueous solution,

[0023] adding further elements to the aqueous solution,

[0024] adding an inoculant including microorganisms
to the aqueous solution,

wherein, by the abovementioned steps, an initial product is
provided, and wherein, subsequently, the following steps are
performed:

[0025] hermetically sealing the container,

[0026] varying a temperature of the initial product and/
or the intermediate products developing therefrom in a
range from 40 to 80° C.,

wherein, after having added the substrate in the first sub-
strate dosage, the following steps for monitoring the cultiva-
tion are performed:

[0027] taking a reference liquid sample and determining
a first concentration of organic substance in the refer-
ence liquid sample,

[0028] taking another liquid sample and determining
another concentration of organic substance in the
further liquid sample afier the expiration of the first
waiting time,

[0029] if the further concentration of organic substance
is smaller than 10 percent of the first concentration of
organic substance, adding substrate in another substrate
dosage,

[0030] repeating the abovementioned steps for monitor-
ing the cultivation until a sufficient amount of biomass
is present in the container.

[0031] Compared to the method in which aerobic micro-
organisms are cultivated, other pH values can be used here.
The value range is 4.5 to 7.5. In the method for the anaerobic

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cultivation, no air is introduced either, but the container in
which the initial product is contained is hermetically sealed.
The temperature for the cultivation of the anaerobic micro-
organisms is selected so as to be higher than in the cultiva-
tion of the aerobic microorganisms. It may be in a range
from 40 to 80° C., preferably in a range of about 55° C.
The other process steps are similar or comparable, which
also applies to numerous of the preferred embodiments of
the method for the aerobic or anaerobic cultivation of micro-
organisms specified below.

[0032] The methods are advantageously further developed
particularly in that a sufficient amount of biomass is present
in the containers when 50 g of wet biomass are extractable
from one litre of product volume, wet biomass having a
moisture content of 93 to 99 percent. Such a sufficient
amount of biomass should generally be reached in the
course of two weeks in the aerobic method while the aerobic
method provides for a sufficient amount of biomass after
about 50 days. Whether the amount is sufficient is deter-
mined by extracting biomass concentrate from the container,
for example 400 ml, and centrifuging this concentrate at, for
example, 4000 revolutions per minute and, for example, 3 to
8° C. The excess obtained in the centrifugation is retuned.
What remains is wet biomass having a moisture content
from 93 to 99 percent, in case of success in an amount of
more than 50 grams per litre of product volume.

[0033] Usefully, it is contemplated that the aqueous solu-
tion is based on deionised water. Since the mineral compo-
sition existing in the initial product may influence the culti-
vation of the microorganisms it is useful to use deionised
water to clearly define the conditions prevailing in the initial
product in this way, particularly on the basis of the addition
of further elements influencing the growth process.

[0034] In particular, the method may be performed on the
basis of using glucose and/or saccharose and/or ethanol and/
or methanol as the substrate.

[0035] It is further contemplated that the first substrate
dosage is added in a concentration of 0.5 g/l/d to 2 g/l/d
and the further substrate dosages are added in a concentra-
tion of 0.5 g/l/d to 2 g/l/d, respectively in relation to the
volume of the aqueous solution. So, when dealing with a
volume of an initial product of one litre, it is, empirically,
useful to add about one gram of substrate per day. Whether
this is the correct amount will of course be verified by the
sample collection within the scope of monitoring the
cultivation.

[0036] For appropriately influencing the cultivation of the
microorganisms, it is further contemplated that microele-
ments and/or macroelements are added as further elements,
the microelements being selected from the group of Mn,
Mo, Zn, Cu, Co, Ni, Cl, Br, and the macroelements being
selected from the group of C, H, O, N, 8, K, Ca, P, Mg, Fe.
Here, microelements are added in lower concentrations, and
macroelements in higher concentrations.

[0037] It is particularly useful that the microelements are
added in a concentration of 0.5 * 10-5 g/l to 2 * 10° g/l and
the macroelements in a concentration of 0.5 g/l to 2 g/l,
respectively in relation to the volume of the aqueous
solution.

[0038] It is preferred that the inoculant contains bacteria
and/or fungi and/or protozoa.

[0039] On this basis it is useful that the inoculant is added
in a concentration of 5 to 10 percent in relation to the
volume of the aqueous solution. The inoculant itself prefer-

May 11, 2023

ably contains water in the range of 90 to 95 percent, the
remaining components being mainly or to a large extent
microorganisms. Of course, it is also possible to add the
microorganisms to the aqueous solution in a signiftcantly
more concentrated form or in a considerably more diluted
form. The indicated ratios have proven useful in practice.
[0040] When performing the method for the aerobic culti-
vation of microorganisms, it is further contemplated that air
is introduced during predetermined periods of time and that
outside of these predetermined periods of time, no air is
introduced. This intermittent supply of air, on the one
hand, establishes the required chemical composition of the
preparation while, on the other hand, in the rest phases, Le.,
when no air is introduced, an unobstructed development of
the cultivation can take place.
[0041] It is contemplated that, during the air supply
phases, the rate of introduced air in relation to one litre of
the volume of the initial product is from | I/min to 2 I/min.
For example, a supply rate of 1.5 I/min has been proven in
practice.
[0042] The substances containing aerobic and/or anaero-
bic microorganisms produced in this way may be used to
produce a preparation for cleaning radioactive liquids and
radioactively charged surfaces. The aerobic and/or anaero-
bic microorganisms may also be obtained from other
sources than via the cultivation methods described above.
[0043] Insofar, the invention consists in a method for the
production of a preparation for cleaning radioactive liquids
comprising the steps of:
[0044] providing a substance including aerobic micro-
organisms, and/or
[0045] providing a substance including anaerobic
microorganisms,
wherein the substance is subjected to the following steps for
washing:
[0046] centrifuging the substance for producing wet
biomass,
[0047] adding an isotonic solution to the wet biomass
for producing a washed substance,
wherein the steps for washing are repeated a plurality of
times, if required, to thereby provide the preparation for
cleaning radioactive liquids and radioactively charged sur-
faces.
[0048] The centrifugation of the substance is preferably
performed at 4000 revolutions per minute at a temperature
of 3 to 8° C. The duration of the centrifugation is usefully 8
to 12 minutes. In particular, the excess of the centrifugation
may be analysed for its composition, particularly its elemen-
tary composition, and utilized for the further cultivation of
biomass. The isotonic solution is preferably used in a ratio
of biomass to solution of 1:4 to 1:10. By sufficiently shaking
or otherwise moving the mixture for preferably 5 to 10 min-
utes, a washed substance without clots is obtained. The solu-
tion or mixture resulting therefrom may, in turn, be centri-
fuged at, for example, 4000 revolutions per minute at 3 to
8°C. for 8 to 12 minutes. Now, the excess may generally be
discarded since it is not to be expected that substantial
amounts of biomass remain for recultivation. The centrifu-
ging and the supply of the isotonic solution may be repeated
a plurality of times, particularly three times in practice.
[0049] According to a specific embodiment, it is contem-
plated that the substance including aerobic microorganisms
and the substance including anaerobic microorganisms are
mixed prior to being washed. Potentially, aerobic microor-

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ganisms and anaerobic microorganisms can coexist. If such
a mixture of microorganisms is desired mixing the aerobic
and the anaerobic microorganisms prior to centrifuging and
washing suggests itself. However, of course aerobic cultures
of microorganisms and anaerobic cultures of microorgan-
isms may also be processed separately.
[0050] According to a particularly preferred embodiment,
it is contemplated that, after washing, an analysis of the
washed biomass is carried out. Depending on the analysis,
the washed biomass can be further processed.
[0051] This may, in particular, be realised so that the pre-
paration for cleaning radioactive liquids and radioactively
charged surfaces is filled into water-permeable cartridges
in portions.
[0052] Furthermore, it is possible that the preparation for
cleaning radioactive liquids and radioactively charged sur-
faces is dried by means of lyophilisation. In this case, the
preparation is available as a dry powder.
[0053] The preparation produced in this way can be used
for cleaning radioactive liquids and radioactively charged
surfaces.
[0054] In particular, the invention consists in a method for
cleaning radioactive liquids comprising the steps of:
[0055] providing radioactive liquid to be cleaned in a
reactor,
[0056] adding a preparation for cleaning radioactive
liquids produced as described above into the reactor,
[0057] adding a substrate into the reactor,
[0058] adding elements selected the from the group of
K, Ca, P, S, N, Zn, Co, Mn, Cl, Cu, Mo, Ni, Se, B, Fe,
wherein, by the abovementioned steps, a working medium
is provided, and wherein, further, the following step is per-
formed:
[0059] removing cleaned radioactive liquid from the
reactor.
[0060] The cleaning of the radioactive liquid is based on
the fact that radioactive isotopes are transferred from the
liquid into the microorganisms, namely by the microorgan-
isms incorporating these radioactive isotopes in their cell
structures while growing. Since the growth the microorgan-
isms and particularly their selectivity with regard to specific
radioactive isotopes can be influenced by adding the indi-
cated elements the cleaning process can be encouraged by
adding these elements. In contrast to the classical chemical
processes, growing microbiological systems can ensure an.
extremely high selectivity and completeness of the extrac-
tion of various chemical elements (among them also radio-
isotopes) from various media. Microorganisms store metals
by accumulation on the surface of the cell or in their interior.
In the process, the storage of metals in the microorganisms
does not only function in case of low metal concentrations in
water solutions, but also in case of extremely high concen-
trations. This storage results in that the metal concentrations
in the biomass of microorganisms can be higher than in the
environment by a plurality of orders of magnitude. One and
the same strain of microorganisms is capable of efficiently
storing metals to be found in various groups of the periodic
system. Such a distinct non-specificity in the storage of
metals by the microorganisms indicates that physicochem-
ical interaction mechanisms prevail between microorgan-
isms and metals. In most cases, the storage of metals takes
place by deposition on the cell surface. The metals may
bond to capsular polysaccharides, a plurality of metals
being stored in one capsule at the same time. The storage

May 11, 2023

of metals on the cell surface may take place by the formation.
of a complex compound of metals and cell membrane pro-
teins. Many metals form inseparable compounds on the cell
surface, for example metal sulphides. The formation of inse-
parable compounds is sometimes also linked to the activity
of ferments. The storage of the metals on the surface of the
microorganisms is also realised by a bond to cell wall struc-
tures. The metals may not only be bound by the cell surface
structures, but also stored in the cell interior. Obviously, the
storage of the metals within the cells is associated with the
function of transport systems. Here, both the active and the
passive transport are involved in the metal extraction. After
the metals have entered the cells, they may bond to cytoplas-
matic proteins and interior membrane structures or form
inseparable products within the cell. Although the metals
are stored in the microorganisms with a clear spatial locali-
sation in most cases, they are sometimes distributed to var-
ious locations, e.g. in the cell wall, in the membrane, and in
the cytoplasm. Compared to the existing methods which
make use of chemical reagents and synthetic sorbent sub-
stances the price of the biological method is many times
lower due to the low costs of the production of the biological
cultures and the facilities required for their use.

[0061] It is advantageous that a selection from the group
of K, Ca, P, S, N, Zn, Co, Mn, Cl, Cu, Mo, Ni, Se, B, Fe of
added elements is depleted of one or more elements which
are to be understood to be biochemical equivalents of the
elements on which the radioactivity of the radioactive liquid
is based. In the selection of the elements to be added, those
which can be referred to as biochemical equivalents of the
elements underlying the radioactive isotopes to be removed
should not be selected. For example, K can be regarded as a
biochemical equivalent of Cs. So, when it is intended to
remove !37Cs from the liquid, no K should be added. Like-
wise, Ca and Sr are to be understood to be biochemical
equivalents.

[0062] Usefully, it is contemplated that one or more liquid
samples are taken from the reactor and tested for their radio-
activity and that, when a maximum value of the radioactiv-
ity is fallen below, the step of removing cleaned radioactive
liquid from the reactor is performed.

[0063] In addition or alternatively, the method is further
developed so that the step of removing cleaned radioactive
liquid from the reactor is performed after the expiration of a
predetermined period of time.

[0064] According to a further development of the method
according to the invention, the approach is that, after the
step of removing cleaned radioactive liquid from the reactor,
the cleaned radioactive liquid is filtered and treated as a non-
radioactive liquid.

[0065] Furthermore, the method is further developed so
that, after the step of removing cleaned radioactive liquid
from the reactor, the biomass is removed from the reactor
and dehydrated as well as subsequently incinerated under
heat supply.

[0066] According to a particularly advantageous embodi-
ment, the approach is that the liquid obtained by the dehy-
dration is tested for its radioactivity, wherein, when a max-
imum value of the radioactivity is fallen below, the liquid is
treated as a non-radioactive liquid, and wherein, when a
maximum value of the radioactivity is exceeded, the liquid
is introduced into one or more earlier process steps in which
the radioactive liquid present there is not or not yet suffi-
ciently cleaned. In this way, a sufficient cleaning of the

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liquid can be achieved within a single procedural setup even.
though this cannot be guaranteed within a single process
step.

[0067] It is advantageous that the working medium is
stirred,

[0068] Particularly in case of purely aerobic microorgan-
isms utilised in connection with the production of the pre-
paration, it is contemplated that air is introduced into the
working medium.

[0069] Similar to the cultivation of the aerobic microor-
ganisms, the approach with regard to the supplied air is
that the rate of introduced air in relation to one litre of the
volume of the working medium is from | 1/min to 2 I/min.
[0070] Usefully, the method is further developed so that a
temperature of the working medium is kept in the range ofa
target temperature, particularly by temperature control.
[0071] It is particularly advantageous that the target tem-
perature is in a range from 25 to 60° C. In case of the use of
aerobic microorganisms, it has proven advantageous that the
temperature is about 23 to 25° C. Otherwise, in case of anae-
robic microorganisms, the temperature may be higher, for
example about 55° C. When aerobic and anaerobic micro-
organisms are mixed a selection or variation between these
thresholds is made.

[0072] Furthermore, it is advantageous that glucose and/or
saccharose and/or ethanol and/or methanol are added as the
substrate.

[0073] In this connection it is advantageous that the sub-
strate is added in a concentration of 0.5 g/l/d to 2 g/l/d in
relation to the volume of the working medium.

[0074] Furthermore, the invention relates to a method for
cleaning a radioactively charged surface in which a prepara-
tion produced as described above is applied to the surface.
Particularly radioactively charged environments of nuclear
power plants are the target of this variant of the invention.
The preparation is applied to the surfaces in dry form, water-
ing the surfaces as required being an option to thereby pro-
mote a distribution on the surfaces or an entry of the pre-
paration into them. The preparation may also be applied ina
dissolved form, particularly in a water solution.

[0075] Therefore, the biological processing of the liquid
radioactive waste is future-compliant both from an ecologic
and from an economic point of view. The present invention
relates to the processing of the liquid radioactive waste of
lower and medium activity including a broad spectrum of
radionuclides. Depending on the type and the properties of
the waste, a biological structure of variable composition is
used, namely adapted microorganism associations com-
posed of bacteria and/or fungi and/or protozoa. The species
composition of the microorganisms in the preparation varies
and may contain up to 3000 or more strains. The effect of
the preparation is based on the fact that life and growth of
the microorganisms are possible in water solutions contain-
ing various salts, among them heavy metal and radionuclide
salts, and having different pH values (from 4.5 to 9.0).
[0076] As is generally known, various chemical com-
pounds dissolved in the water which are referred to as
macro- and microelements here are required for the growth
and reproduction of the microorganisms. The macroele-
ments include: C, H, O, N, S, K, Ca, P, Mg, Fe. The micro-
elements such as Mn, Mo, Zn, Cu, Co, Ni, Cl, Br, ete. are
needed by the microorganisms in trace amounts. The biolo-
gical processing of the liquid radioactive waste takes place
during the growth of the microorganisms of the preparation

May 11, 2023

in water solutions of the liquid radioactive waste. In the pro-
cess, the microorganisms are activated by a rational change
in the element composition in the liquid radioactive waste,
the change in the ambient temperature, the breathing condi-
tions (aerobic-anaerobic conditions), the introduction of
specific substrates and other macro- and microelements.
The rational change of the element composition in the med-
ium means an exclusion of elements urgently required for
the microorganism activity. Here, the radionuclides in the
liquid radioactive waste have to function as biochemical
equivalents of the excluded elements. In case of the biolo-
gical processing of the liquid radioactive waste, elements
having similar ion radii are to be regarded as biochemical
equivalents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The invention will now be explained by way of
example with reference to the accompanying drawings
with the aid of particularly preferred embodiments.

[0078] FIG. 1 shows a diagram for illustrating a method
for the aerobic cultivation of microorganisms;

[0079] FIG. 2 shows a diagram for illustrating a method
for the anaerobic cultivation of microorganisms;

[0080] FIG. 3 shows a diagram for illustrating of a method
for cleaning radioactive liquids;

[0081] FIG. 4 shows a flow diagram for explaining a
method for cleaning radioactive liquids.

DETAILED DESCRIPTION OF THE INVENTION

[0082] FIG. 1 shows a diagram for illustrating a method
for the aerobic cultivation of microorganisms. For the aero-
bic cultivation von microorganisms, an aqueous solution 10
preferably having a pH value of 5.5 to 9.0 is provided in a
container 12. The aqueous solution 10 is based on deionised
water. To the aqueous solution 10, a substrate 14, particu-
larly glucose is added. In addition, further elements are
added, namely microelements and/or macroelements, the
microelements being selected from the group of Mn, Mo,
Zn, Cu, Co, Ni, Cl, Br, and the macroelements being
selected from the group of C, H, O, N, 8, K, Ca, P, Mg,
Fe. Likewise, an inoculant 18 forming the basis of the bio-
mass 20 to be cultivated is added. Outside of the container
12, a compressor 32 is provided which can introduce air into
a jet nebuliser 36 through a pipe 34. The air is transported to
the bottom of the container 12 so that it can be directly
applied to the biomass 20 mainly present on the bottom of
the container 12 there. Furthermore, a heating device 38 is
disposed outside of the container 12. The heating device 38
is connected to a thermocouple 42 via a temperature control-
ler 40 so that the temperature of the substances present in the
container 12 can be controlled and/or regulated. Likewise,
biomass collectors 44 are provided by means of which bio-
mass 20 can be collected from the container 12.

[0083] FIG. 2 shows a diagram for illustrating a method
for the anaerobic cultivation of microorganisms. In the con-
tainer 12' for the anaerobic cultivation of microorganisms,
again, an aqueous solution 10’ is contained, however, pre-
ferably having a pH value of 4.5 to 7.5. To the aqueous solu-
tion 10’, again, a substrate 14’, preferably glucose, other ele-
ments 16’, namely microelements and macroelements as
indicated above, and an inoculant 18’ are added. Here as
well, the inoculant 18’ is the initial substance for the bio-
mass 20’ cultivated therefrom. Also, again a heating device

Page 11

US 2023/0143790 Al

38’ is provided which is connected to a thermocouple 42' via
a temperature controller 40’, the heating device 38' and the
temperature controller 40' being disposed outside of the
container 12' while the thermocouple 42' is disposed in the
container 12’ and particularly in the substances present in
the container 12’ so that again the temperature of the sub-
stances can be controlled and/or regulated. The container 12'
further contains one or more biomass collectors 44' for col-
lecting biomass 20’. The container 12’ is hermetically closa-
ble by means of a closure 46’, particularly to prevent air
from entering. In order to render the discharge of gases
released within the container 12’ possible, a siphon-like
water trap 48' is provided on the upper side of the container
12’.

[0084] The preparation which is ultimately to be used for
cleaning radioactive liquids and which is obtained on the
basis of the described cultivation of microorganisms con-
tains various groups of aerobic and anaerobic microorgan-
isms, among them extremophiles which can exist under
extreme conditions, namely at low and high temperatures
(psychrophilies and thermophilies), in a high salt content
(hallophilies) as well as at high radiation levels (radioresis-
tant microorganisms).

[0085] The preparation of the preparation takes place by
the non-sterile cultivation of various microbial primal asso-
ciations originating from natural and technogenic sources by
varying the growth conditions with the aim of obtaining
associations characterised by a mutually beneficial coexis-
tence. In addition, the climatic conditions of the intended
place of use as well as the background composition of the
primal microorganisms are taken into account in the pre-
paration of the preparation. During the cultivation, apart
from biogenic compounds, also such compounds which are
toxic for microorganisms - in doses from harmless to semi-
lethal - are contained in the solutions for the cultivation of
the preparation. In addition, the solutions may contain the
following substances: salts of heavy metals, petroleum pro-
ducts (among them polyaromatic hydrocarbons), surfac-
tants, carbohydrates, protein, fats, etc. In an aggressive
environment, non-surviving microorganisms of the prepara-
tion are a source for additional protection and nutrition for
the surviving community.

[0086] A cultivation method according to the invention as
the basis for the production of the preparation for cleaning
radioactive waste can also be described as follows.

[0087] A first process step is the extraction of liquid (with-
out biomass) on the occasion of the first addition of | ml
substrate as well as the subsequent centrifugation at
14500 rpm for 3 minutes at ambient temperature. A content
analysis is performed on the organic substance.

[0088] A second process step is the extraction of liquid
(without biomass) 24 hours after the addition of 1 ml sub-
strate including the subsequent centrifugation at 14500 rpm.
for 3 minutes at ambient temperature. A content analysis is
performed on the organic substance.

[0089] Provided that, in the third process step, it is found
that 90% of the organic substance was consumed, the next
substrate dosage should be added within the course of
14 days (aerobic) or 50 days (anaerobic), regular content
analyses of the organic substance and of the biogenic ele-
ments having to be carried out. In case there was no con-
sumption of 90 percent the second process step is repeated.
[0090] Otherwise, the collection of 400 ml of biomass
concentrate from a bioreactor as well as a centrifugation of

May 11, 2023

the concentrate at 4000 rpm at 3 to 8° C. will be effected in
the fourth process step (production stage). The excess of the
centrifugation is retuned into the bioreactor. The mass of
moist biomass is determined, likewise the moisture content
of the biomass.

[0091] Ina fifth process step, the adapted biomass is ready
for use if the mass of the centrifuged wet biomass is more
than 50 grams per litre. Otherwise, the third process step and
the following are repeated.

[0092] When the microbial association which, in particu-
lar, can be obtained by the described cultivation methods is
thus ready for use compounds impeding the intended pro-
cess are extracted from the biomass. The biomass is sepa-
tated from the solution by centrifugation. If required, the
biomass cultivated in various modes is mixed and washed
three times with the isotonic solution on the basis of deio-
nised water such as, for example, 0.1 to 0.9% NaCl solution
in the ratio of 1:10 to 1:30. Then, it is centrifuged. The
essence of this method is that the washing of biomass
extracts compounds impeding the intended process of pro-
cessing liquid radioactive waste.

[0093] A washing method within the scope of the produc-
tion of the preparation for cleaning radioactive waste can
also be described as follows.

[0094] From the containers in which the biomass is culti-
vated in the aerobic and anaerobic mode, the culture liquids
are extracted and mixed in the ratio of 1:5 to 5:1 as well as
centrifuged at 4000 revolutions per minute at a temperature
of 3 to 8° C. for 8 to 12 minutes. The excess water is ana-
lysed for its elementary composition and introduced into a
container for the recultivation of biomass.

[0095] The obtained wet biomass is added into isotonic
solution in the ratio of biomass to solution of 1:4 bis 1:10
and thoroughly agitated for 5 to 10 minutes to avoid or elim-
inate a formation of clumps of the biomass.

[0096] The obtained solution is centrifuged at 4000 rpm at
3 to8° C. for 8 to 12 minutes. The excess water is discharged
into the sewage system. The process is repeated, for exam-
ple, three times.

[0097] In order to verify that the undesired elements were
completely removed from the biomass, mass spectrometric
analyses for the presence of elements are carried out on the
initial excess and the third washing solution.

[0098] The washed moist biomass constituting the basis of
the preparation for cleaning radioactive liquids is used for
the processing of liquid radioactive waste either in a free
form or in a special container or cartridge.

[0099] Special cartridges are hermetically closed contain-
ers made of a water-permeable non-woven fabric which let
no microorganisms escape to the outside.

[0100] There is also the option to have the preparation pre-
pared in the form of a dry powder, namely by lyophilisation.
The approach for obtaining a dry powder including about
90% viable microorganisms is as follows.

[0101] Into the moist, washed, (after centrifuging) paste-
like biomass, a cryoprotector is introduced, e.g. a water
solution of dextran polysaccharide - 40% polyglucan solu-
tion in the ratio of 2.5% polyglucin in the finished paste and
2.5% glycerin (for 70 I of paste - 4.37 I of the 40 percent
polyglucan solution and 1.75 I of glycerin). It is mixed for
10 minutes.

[0102] The finished mixture is frozen at -70° C.

Page 12

US 2023/0143790 Al

[0103] The frozen mixture is subjected to lyophilic dehy-
dration until a powder having a residual moisture of not
more than 1 - 3% is formed.

[0104] This powder is tightly sealed in polyethylene bags
under vacuum.

[0105] The properties of the preparation in the dry form
remain unchanged for 3 years.

[0106] Therefore, the finished preparation consists of sev-
eral thousands of microorganisms of various species
adapted to life in hash conditions. Part of the microorgan-
isms is capable of developing in aerobic and anaerobic con-
ditions at a temperature of 20 to 80° C.

[0107] The cultivation and further processing of the
microorganisms for the preparation does not require sterile
conditions, expensive devices and reagents which renders
keeping the original costs of the production of the prepara-
tion low possible.

[0108] FIG. 3 shows a diagram for illustrating a method
for cleaning radioactive liquids. A primary container 50 is
provided in which radioactive liquid 24 to be cleaned is pre-
pared. The radioactive liquid 24 to be cleaned is introduced
into the container 50 through an opening 52 provided on the
upper side of the primary container 50, along with, if
required, deionised water 54 and macro- and microelements
56 as listed in detail above. The mixture or solution present
in the primary container 50 is supplied to an analysis unit 58,
it being decided based on the results of the analysis whether
the mixture or solution is to be changed in its composition,
for example to be diluted. As soon as the content of the
primary container 50 has the desired properties, the content
is completely or partly removed and supplied to a conveyor
system 60. This conveyor system 60 conveys the content,
i.e. the radioactive liquid 24 to be cleaned, into the reactor
22. Apart from the radioactive liquid 24 to be cleaned, a
preparation 28 which was, particularly, obtained on the
basis of the cultivation described in connection with FIGS.
1 and 2, as well as again a substrate 30, particularly glucose,
are supplied to the reactor 22. Outside of the reactor 22, a
heating device 62 is provided which is connected to a ther-
mocouple 66 placed in the reactor 22 via a temperature con-
troller 64 also disposed outside of the reactor 22. Thus, the
temperature in the reactor 22 or of the substances present in.
the reactor 22 can be controlled and/or regulated. The reac-
tor 22 is hermetically closable by a closure 68. In order to
allow for gases released within the reactor 22 to be dis-
charged, a siphon-like water trap 70 is provided on the
upper side of the reactor 22. For the purpose of the air sup-
ply, a compressor 72 connected to a jet nebuliser 76 within
the container via a pipe 74 is disposed outside of the reactor
22. Furthermore, an agitator 78 by means of which the sub-
stances in the reactor 22 can be stirred is disposed in the
reactor 22. The content of the reactor 22 can be supplied to
an analysing unit 98. Essentially, the reactor 22 is capable of
processing aerobic and anaerobic microorganisms to clean
the added radioactive liquid 24 based on them in this way. In.
case of a strictly anaerobic biomass, however, the means for
supplying air, i.e. the compressor 72, the pipe 74, and the jet
nebuliser 76, can, in principle, be omitted. The reactor 22
further comprises a biomass discharge system 80 through
which the biomass 28 is suppliable to a filter press 82. In
this filter press 82, the biomass 28 can be mechanically
dehydrated, whereupon it is suppliable to a heating plate
84 to be incinerated there. Depending on whether the ash
is below a radioactivity threshold, it can be supplied to a

May 11, 2023

disposal 86 as low risk waste, or it is disposed of in a final
disposal site 88 for the final storage of radioactive waste.
The liquid emerging from the filter press 22 is supplied to
an analysing unit 90. In this analysing unit, it will be deter-
mined whether the liquid is below a radioactivity threshold.
If this is the case the liquid can be supplied to a waste water
container 92 in which cleaned radioactive liquid 26, i.e. par-
ticularly no longer radioactive liquid or modestly radioac-
tive liquid, from the filter press 82 is collected. However,
if the radioactivity of the liquid from the filter press 82
exceeds a predetermined radioactivity threshold, it is
retumed to one of the preceding process stages, i.e., in par-
ticular, supplied to the primary container 50 or the reactor
22. The waste water container 92 is also supplied with the
liquid freed from radioactivity from the reactor 22, namely
through the reactor’s on-site discharge system 94 for
cleaned liquid as well as a coarse cleaning filter 96.

[0109] FIG. 4 shows a flow diagram for explaining a
method for cleaning radioactive liquids. In step S01, radio-
active liquid is made available in a primary container. The
content of the primary container is analysed in step S02, a
change of the content depending on the results of the analy-
sis still being possible, for example by the targeted addition
of macroelements and/or microelements and/or of additional
deionised water for dilution. In step $03, the radioactive
liquid is filled into the reactor. In step $04, the preparation
for cleaning is supplied, and in step S05 the substrate, i.e.
particularly glucose. Subsequently, according to step S06,
there is a waiting time of a period of time t. In step S07, a
liquid sample is taken and analysed for radioactivity. If, in
step $08, it is determined that the radioactivity (RA) has not
yet fallen below a maximum radioactivity (RAmax), the
course of the process will be retumed to step S06, and
there is another specified waiting time. However, when the
radioactivity has fallen below a maximum radioactivity
(RAmazx), the liquid can be removed from the reactor. It is
filtered in step $14, and filled into a container for non-radio-
active waste water in step $15. The biomass is also removed
from the reactor and dehydrated in step S09. The dehydrated
biomass is incinerated in step $12 and permanently stored in
step S13. Instead of a final storage, it can also be disposed of
as low risk waste in case of a sufficiently low or no longer
detectable radioactivity of the incinerated biomass. The
liquid resulting from the dehydration performed in step
S09 is analysed for its radioactivity in step S10. In step
S11, it is tested whether the radioactivity (RA) is smaller
than a maximum radioactivity (RAmax). If this is the case
the liquid can be filled into a container for non-radioactive
waste water according to step S15. If the radioactivity (RA)
is not lower than a maximum radioactivity (RAmax) the
liquid is retumed to the primary container according to
step $17 or to the reactor according to step $16.

[0110] For cleaning purposes, the radioactive waste is thus
introduced into a special reactor enabling a thermostat func-
tion for its content as well as ventilation and/or stirring with
air and/or an agitator. In the reactor, there is also the prepara-
tion, a set of macro- and microelements and a substrate for
the growth of the culture. The process takes place at a tem-
perature of 20 to 80° C. In case of aerobic cultures, flowing
air from an external compressor is supplied through a tube
having a weight and a nebuliser at the outlet at a rate of
1.5 bis 3.0 l/min per one litre of the liquid radioactive
waste for ventilation. The duration of the ventilation and/
or stirring varies from continuously to periodically depend-

Page 13

US 2023/0143790 Al

ing on the processing objective. For example, the duration.
may be 20 s per day. In the process, the preparation should
be stirrable from the bottom to the surface; however, a leak-
age of the biomass on the upper container walls (not covered
by liquid) in the course of the method is to be avoided. The
consumption of the preparation varies from | bis 35 g moist
biomass (moisture 93 - 99%) or 0.01 - 2.45 g lyophilised
powder per one litre of the liquid radioactive waste. As the
substrate, for example, highly pure glucose is used which is
added in an amount of 0.5 to 5 g per one litre of the liquid
radioactive waste per day. As the biogenic elements, K, Ca,
P, S,N, Zn, Co, Mn, Cl, Cu, Mo, Ni, Se, B, Fe are intro-
duced. However, to achieve the conditions of an accelerated
biosorption, particularly in case of anaerobic cultures, or to
reduce the activity the solution, particularly in case of aero-
bic cultures, one or some elements should be missing in the
medium to be processed if they are stoichiometric equiva-
lents of the radionuclide to be processed which is used for
growth by the microorganism cells.
[0111] Depending on the processing objectives, the liquid
radioactive waste is converted into solid radioactive waste
with a multiple reduction of volume, or the activity of the
liquid radioactive waste is directly reduced.
[0112] The liquid radioactive waste is first supplied to a
preparation container or primary container. Samples are
taken to carry out necessary analyses, e.g. with the follow-
ing methods:
[0113] mass spectrometry (identification of stable and
radioactive elements);
[0114] ion chromatography by means of a conducto-
metric detector;
[0115] potentiometry.
[0116] The effect of the preparation was, in particular,
tested under the following conditions:
[0117] radioactivity of the solution lower than
2-10? kBq/l;
[0118] overall salt content lower than 100 g/l;
[0119] pH value of the medium from 5 to 9.
[0120] After having performed the examinations, it is
therefore recommended to dilute the liquid radioactive
waste with deionised water when required. Then, macro-
and microelements are introduced into the primary container
when required.
[0121] It is important to emphasise that the liquid radio-
active waste should not contain any stable isotopes of the
radionuclides to be disposed of so as to be capable of suc-
cessfully reducing the radioactivity. For growth, the micro-
organisms will, first of all, absorb the stable isotopes of the
radionuclides to be disposed of. From the mixture !33Cs and
137Cs, ¢.g., the preparation will first process the stable iso-
tope 133Cs. Consequently, the radioactivity of the liquid
radioactive waste will not change in the process.
[0122] The prepared liquid radioactive waste is supplied
into the reactor via the conveyor system.
[0123] Then, the preparation and the substrate for the
growth of the microbiological cultures are added into the
reactor. While the ventilation and/or stirring are deactivated,
the reactor lid is tightly closed. The lid is equipped with a
liquid seal filled with deionised water and discharging
excess gas from the reactor when required. The temperature
in the reactor is maintained at the target level by means of a
heating element and a temperature control encoder. The
temperature of the medium in the reactor is adjusted with
the aid of a thermocouple. The ventilation takes place with

May 11, 2023

oxygen from the ambient air which is supplied via the com-
pressor and the jet nebuliser. The stirring is carried out by a
built-in propeller. Regularly, a sample is taken from the
upper liquid layer to determine the radioactivity. When it
is the objective to rapidly store the radionuclides in the
microorganisms for further final storage the maximum bio-
sorption is reached within | to 7 days. If itis the objective to
reduce the radioactivity of the solution while no final storage
of the radioactive substances is intended it is required to
maintain the fast growth of the microorganisms until the
radioactivity is reduced to the background or target level.
[0124] When the objective of cleaning the water solution
from the radionuclides is reached the liquid cleared of the
tadionuclides flows into the containers for the non-radioac-
tive waste water via the discharge system and a coarse filter,
e.g. a sand filter, and the spent biomass of the preparation is
transferred onto the filter press by the biomass-discharge
system. If a cartridge is used the cartridge including the
spent biomass is removed from the water, then mechanically
pressed out above the water surface according to the tea bag
principle, dried in air, and then opened to place the content
(spent biomass) on the heating plate and to bypass the filter
press.

[0125] If the preparation is used in a loose form (without a
cartridge) the spent biomass having a moisture of about 93 -
99% is mechanically dehydrated in the filter press to the
largest possible extent and then placed on the heating plate
for a slight incineration in air at a temperature of not more
than 250° C. In the process, the liquid separated from the
biomass in the filter press is examined for radioactivity. If
the radioactivity of the filtrate does not exceed the target
values the liquid is also discharged into the containers for
the non-radioactive waste water. Otherwise, the liquid flows
back into the reactor or into the primary container for the
preparation of the liquid radioactive waste to be processed
with the next batch of the liquid radioactive waste. If a
reduction of the radioactivity below specific target values
is not required or if no further batch of liquid radioactive
waste is in the queue for cleaning the radioactive filtrate
the volume of which does not exceed the volume of the
spent moist biomass may be evaporated onto the heating
plate for the further final storage of the residual salts.
[0126] When the biosorption of the radionuclides is
applied the generated ash including the radionuclides stored
in it is forwarded for final storage. In case of a reduction of
the radioactivity, the ash poses no risk to humans, plants,
and animals and can be disposed of according to traditional
methods.

[0127] When the objective of clearing the water solution
of the radionuclides by sorption was not reached in the first
application of the preparation it is required to replace the
spent preparation by new preparation. Here, the liquid radio-
active waste and the spent biomass are processed as
described above. The method of biosorption should be
repeated until the radioactivity of the solution is reduced to
the background or target level.

[0128] In the final stage of the processing of the liquid
radioactive waste by means of the method of biosorption
by means of the preparation, low risk “technical” water
and completely dry ash of the microorganisms including
the radioactive elements stored in it are generated. In this
way, a multiple reduction of the amount of waste for final
storage is achieved. In case of a successful transformation of

Page 14

US 2023/0143790 Al

the radionuclides into stable elements, no final storage of the
waste is required.

[0129] The association of microbes existing on the edge of
survival incorporates radionuclides into the cells. In the pro-
cess, the microorganisms grow by lysis of some cells and
discharge of remaining amounts of deficiency macroele-
ments into the water medium. In the biological systems,
the atomic transformations take place in nanoscale spaces
of the microorganism cells. For the protons, the nanoscale
spaces of the growing biological cells represent potential
wells having dynamically changing walls creating coherent
correlated states of the quantum particles. The protons exist-
ing in these states are capable of a reaction resulting in the
formation of elements required for the further performance
of biochemical processes in the microorganisms. Therefore,
the active division of the cells including the radionuclides in
the nanoscale spaces results in the reduction of the radio-
activity under formation of the stable elements from the
radionuclides.

[0130] In the present context, the phenomenon of the
transmutation of atomic nuclei by nucleosynthesis may
play a role. For nucleosynthesis, a number of requirements
has to be fulfilled. One of them is associated with the neces-
sity of incorporating the synthesising isotope in the metabo-
lism as fast as possible including the associated fixation as a
stable nucleus (stable atom). It is obvious that the biochem-
ical processes supporting storage simultaneously promote
the transmutation of atomic nuclei. During the growth of
microbiological cultures, the reproduction of DNA mole-
cules and some other macromolecules, their orientation,
and the formation of the primary and secondary structures
take place. In the area of the structural adaptation, a contin-
uous process of the formation and modification of microin-
homogenities with a characteristic magnitude of the same
order takes place which is required for the “elimination” of
the Coulomb barrier in the transmutation of atomic nuclei. If
the nutrient medium for the microbiological culture does not
contain an isotope of the element required for the growth of
the culture, but the other isotopes which may constitute the
isotope required for the growth as result of the transmutation.
of atomic nuclei this isotope is, upon formation as the result
of a reaction, immediately absorbed by and fixed in the
microbiological culture in optimum micro activities. There-
fore, there is the possibility to utilise the transformation of
the radioactive isotopes absorbed by the bacteria culture into
stable isotopes of other chemical elements. In this case, this
does not refer to a comprehensive use of microbiological
systems in the first stages of the processing of spent fuel
elements and highly active waste.

[0131] In the following, some examples for illustrating the
invention are provided:

[0132] Example 1: As a model for waste water including
heavy metals, deionised water in a volume of 75 ml with
K,Cr.0, dissolved therein in a concentration of 0.1 g/l and
with an overall salt content of about 100 g/l was used. Intro-
duced into the reactor were: A set of macro- and microele-
ments with the exception of biochemical chromium equiva-
lents, then moist paste-like preparation in an amount of
2.21 gas well as a substrate in the ratio of 5 to 6 g organic
substance per one litre of the mixture. The experiment was
predominantly carried out under microaerophilic conditions
at a temperature of 55 to 80° C. In the course of the experi-
ment, the pH value of the medium changed in the range of
6.9 to 7.9. The consumption level of the organic substance

May 11, 2023

and the relevant macro- and microelements were monitored.
Within a maximum of 7 days, the initially coloured solution.
became completely colourless. Spectrophotometric exami-
nations still revealed traces of chromium in the solution.
[0133] Example 2: Here, the approach was like in Exam-
ple | with the exception that the compound K3Cr,07 was
used in a concentration of 10 g/l. In this case, the solution
became colourless within 50 days of the experiment. Spec-
trophotometric examinations again revealed traces of chro-
mium in the solution. The preparation had a bluish tint.
[0134] Example 3: As a model for liquid radioactive
waste, deionised water in a volume of 75 ml with radioac-
tive caesium having an activity of 180 kBq/l dissolved in the
water was used. Introduced into the reactor were: A set of
macro- and microelements with the exception of biochem-
ical caesium-equivalents, then moist paste-like preparation
in an amount of 2.5 g as well as a substrate in the ratio of 3 to
4 g organic substance per one litre of the mixture. The
experiment was predominantly carried out under microaer-
ophilic conditions at a temperature of 55 to 65° C. In the
course of the experiment, the pH value of the medium chan-
ged in the range from 5.4 to 7.5. Within 20 days, the activity
of the liquid was reduced down to 20 kBq/1.

[0135] Example 4: As a model for liquid radioactive
waste, deionised water in a volume of 750 ml with the com-
pound CsNO; in a concentration of 0.5 g/l dissolved in the
water was used. Introduced into the reactor were: A set of
macro- and microelements with the exception of biochem-
ical caesium-equivalents, then moist paste-like preparation
in an amount of 9.55 g as well as a substrate in the ratio of
0.5 bis 1 g organic substance per one litre of the mixture.
The experiment was predominantly carried out under aero-
bic conditions at a temperature of 20 to 28° C. In the course
of the experiment, the pH value of the medium changed in
the range from 6.1 to 7.9. The consumption level of the
organic substance and the relevant macro- and microele-
ments were monitored. Within a maximum of 7 days, mass
spectrometric analyses revealed a transition from Cs into the
biomass. The caesium content in the biomass was up to
300 mg/g of the completely dry biomass.

[0136] Example 5: As a model for liquid radioactive
waste, deionised water in a volume of 75 ml with the radio-
active caesium-compound CsNO; dissolved in the water
was used which guarantees a caesium content at a level of
5-104 Bg. Introduced into the reactor were: A set of macro-
and microelements with the exception of biochemical cae-
sium equivalents, then moist paste-like preparation in an
amount of 0.9 g. No substrate was introduced into the bior-
eactors. The experiment was predominantly carried out
under aerobic conditions at a temperature of 20 to 25° C.
In the course of the experiment, the pH value of the medium
changed in the range from 6.1 to 7.2. The biomass was
removed from the reactor every 1 to 3 days. Radiospectro-
scopic examinations of the biomass revealed a caesium con-
tent in the moist biomass of up to 17 kBq per one gram of
the completely dry biomass.

[0137] Example 6: As a model for liquid radioactive
waste, deionised water in a volume of 700 ml with a com-
pound of stable caesium CsNO; in a concentration of 0.12 g/
I dissolved in the water was used. The preparation was intro-
duced into the reactor so that the bioreactor contained 10 g
of the moist biomass and a set of macro- and microelements
with the exception of biochemical caesium equivalents as
well as a substrate in the ratio of 2 to 3 g organic substance

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US 2023/0143790 Al

per one litre of the mixture. The experiment was predomi-
nantly carried out under microaerophilic conditions at a
temperature of 35 to 45° C. The best result was already
achieved after 8 days of the experiment. An analysis of the
content of the bioreactor by means of atomic emission spec-
trometry with an inductively coupled plasma revealed that
the caesium content was reduced by 55%. In the bioreactors,
barium was detected which may indicate a transmutation of
atomic nuclei.

[0138] Example 7: As a model for liquid radioactive
waste, deionised water in a volume of 750 ml with the radio-
active caesium compound CsNO; dissolved in the water
was used which guarantees a caesium content on a level of
104 Bq. Introduced into the reactor were: A set of macro-
and microelements with the exception of biochemical cae-
sium equivalents, then moist paste-like preparation in an
amount of 9.05 g as well as a substrate in the ratio of 0.5
bis 1 g organic substance per one litre of the mixture. The
experiment was carried out under aerobic conditions at a
temperature of 20 to 28° C. In the course of the experiment
the pH value of the medium changed in the range from 6.5 to
8.1. The consumption level of the organic substance and the
relevant macro- and microelements were monitored. Within
a maximum of 15 days, radiospectroscopic examinations
revealed an average reduction of the activity of the content
of the bioreactor by 23%.

[0139] If radionuclides from water solutions are to be
rapidly concentrated the preparation seems to be a universal
biosorbent which is easy to dispose of and which is capable
of extracting all substances in arbitrary quantity ratios from
the liquid radioactive waste. In case of a rational change of
the element composition in the medium and during the
growth of the microorganisms, the preparation is capable
of transforming predetermined elements and thus transform
the radioactive elements from the liquid radioactive waste
into non-radioactive elements.

[0140] In the biological processing of liquid radioactive
waste under application of the preparation, no extreme tem-
peratures or pressures are required so that the method
requires a low energy intensity. Furthermore, the application
of the preparation does not require the introduction of hazar-
dous chemical reagents which excludes the risk of a second-
ary contamination. The features described above guarantee
utmost simplicity and operational safety of the procedural
facility.

[0141] The features of the invention disclosed in the above
description, in the drawings as well as in the claims may be
important for the realisation of the invention both individu-
ally and in any combination.

List of numerals

10 Aqueous solution
10" Aqueous solution
2 Container

2 Container

4 Substrate

WV Substrate

16 Other elements
16 Other elements
18 Inoculant

18" Inoculant

20 Biomass

20" Biomass

2 Reactor

May 11, 2023

-continued,

List of numerals

24 Radioactive liquid

6 Radioactive liquid

28 Biomass

30 Substrate

2 Compressor

34 Pipe

36 Jet nebuliser

38 Heating device

38" Heating device

40 Temperature controller
40" ‘Temperature controller
2 ‘Thermocouple

a Thermocouple

44 Biomass collector
44 Biomass collector

46° Closure

48" Water trap

50 Primary container

52 Opening

58 Analysis unit

60 Conveyor system

62 Heating device

64 Temperature controller
66 Thermocouple

68 Closure

a Water trap

n Compressor

14 Pipe

76 Jet nebuliser

8 Agitator

80 Biomass discharge system
82 Filter press

84 Heating plate

86 Disposal

88 Final disposal site

90 ‘Analysing unit

92 Waste water container
94 Discharge system

96 Coarse cleaning filter
98 ‘Analysing unit

1. Amethod for the aerobic cultivation of microorganisms,
comprising the steps of:

providing an aqueous solution having a pH value of 5.5 to
9.0 ina container,

adding a substrate ina first substrate dosage to the aqueous
solution,

adding further elements to the aqueous solution,

adding an inoculant with microorganisms to the aqueous
solution,

wherein an initial product is provided by the abovemen-
tioned steps, and wherein, subsequently, the following
steps are performed:

introducing air into the initial product or into intermediate
products developing therefrom,

varying a temperature of the initial product or the inter-
mediate products developing therefrom in a range from
15 to35°C.,

wherein, after having added the substrate in the first sub-
strate dosage, the following steps are performed for mon-
itoring the cultivation:

taking a reference liquid sample and determining a first
concentration of organic substance in the reference
liquid sample,

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US 2023/0143790 Al

taking another liquid sample and determining another con-
centration of organic substance in the further liquid sam-
ple after the expiration of the first waiting time,
if the further concentration of organic substance is smaller
than 10 percent of the first concentration of organic sub-
stance, adding substrate in another substrate dosage,

repeating the abovementioned steps for monitoring the cul-
tivation until a sufficient amount of biomass is present in
the container.

2. (canceled)

3. The method according to claim 1, wherein, a sufficient
amount of biomass is presentin the container when 50 gof wet
biomass are extractable from one litre of product volume, wet
biomass having a moisture content of 93 to 99 percent.

4. The method according to claim 1, wherein the aqueous
solution is based on deionised water.

5. The method according to claim 1, wherein glucose or
saccharose or ethanol or methanol are used as the substrate.

6. The method according to claim 1, wherein the first sub-
strate dosage is added in a concentration of 0.5 g/l/dto 2 g/I/d,
claim 1, and the further substrate dosages are added in a con-
centration of 0.5 g/I/d to 2 g/I/d, respectively in relation to the
volume of the aqueous solution.

7. The method according to claim 1 wherein microelements
or macroelements are added as further elements, wherein the
wherein the microelements are selected from the group of Mn,
Mo, Zn, Cu, Co, Ni, Cl, Br, and wherein the macroelements
are selected from the group of C, H, O, N, 8, K, Ca, P, Mg, Fe.

8. The method according to claim 6, wherein the microele-
ments are added in a concentration of 0.5 * 10-5 g/l to 2 ¥ 10-
5 g/land the macroelements ina concentration of 0.5 g/lto2 g/
1, respectively in relation to the volume of the aqueous
solution.

9. The Method according to claim 1 wherein the inoculant
contains bacteria claim 1, or fungy or protozoa.

10. Themethod according to claim 1, wherein the inoculant
is added in a concentration of 5 to 10 percent in relation to the
volume of the aqueous solution.

11. The method according to claim 1, wherein air is intro-
duced during predetermined periods of time and that no air is
introduced outside of these predetermined periods of time.

12. The method according to claim 11, wherein the rate of
introduced airinrelation to one litre ofthe volume of the initial
product is from 1 1/min to 2 l/min.

13. A method for the production ofa preparation for clean-
ing radioactive liquids and surfaces, comprising the steps of:

providing ofa substance with aerobic microorganisms, cul-

tivated using a method aerobic of cultivation of microor-
ganisms comprising the steps of:

providing an aqueous solution having a pH value of 5.5 to

9.0 ina container.

adding a substrate in a first substrate dosage to the aqueous

solution,

adding further elements to the aqueous solution,

adding an inoculant with microorganisms to the aqueous

solution,

wherein an initial product is provided by the abovemen-

tioned steps, and wherein, subsequent, the following
steps are performed:

introducing air into the initial product or into intermediate

products developing therefrom,

varying a temperature of the initial product or the inter-

mediate products developing therefrom in a range from
15 to 35 degree Celsius,

May 11, 2023

wherein, after having added the substrate in the first sub-
strate dosage, the following steps are perfomed for mon-
itoring the curtivation:

taking a reference liquid sample and determining a first

concentration of organic substance in the reference
liquid sample.

taking another liquid sample and determining another con-

centration of organic substance in the further liquid sam-
ple after the expiration of the first waiting time,
if the further concentration of organic substance is smaller
than 10 percent of the first concentration of organicsub-
stance, adding substrate in another substrate dosage.

repeating the abovementioned steps for monitoring the cul-
tivation until a sufficient amount of biomass is present in
the container, or
providing of a substance with anaerobic microorganisms,
particularly cultivated according to one of the claims 2 to
10 cultivated using a method for the anaerobic cultiva-
tion of microorganisms, comprising the steps of:

providing an aqueos solution having a pH value of 4.5 to7.5
ina container

adding a substrate ina first substrate dosage to the aqueous

solution,

adding further elements to the aqueous solution,

adding an inoculant with microorganisms to the aqueous

solution,

wherein an initial product is provided by the abovemen-

tioned steps, and wherein, subsequently, the following
steps are performed:
hermetically sealing the container,
varying a temperature of the initial product or the inter-
mediate products developing therefrom in a a range
from 40 to 80° C.,

wherein, after having added the substrate in the first sub-
strate dosage, the following steps are performed for mon-
itoring the cultivation:

taking a reference liquid sample and determining a first

concentration of organic subtance in the reference liquid
sample,

taking another liquid sample and another concentration of

organic substance in the further liquid sample after the
expiration of the first waiting time,
if the further concentration of organic substance is smaller
than 10 percent of the first concentration of organic sub-
stance, adding substrate in another substrate dosage,

repeating the abovementioned steps for monitoring the cul-
tivation until a sufficient amount of biomass is present in
the container,

wherein the substance is subjected to the following steps for

washing:

centrifuging the substance for producing wet biomass,

adding an isotonic solution to the wet biomass for produ-

cing a washed substance, wherein the steps for washing
are repeated a plurality of times, if required, to thereby
provide the preparation for cleaning radioactive liquids
and surfaces.

14. The method according to claim 13, wherein the sub-
stance with aerobic microorganisms and the substance with
anaerobic microorganisms are mixed prior to being washed.

15. The method according to claim 13, wherein, after wash-
ing, an analysis of the washed biomass is carried out.

16. The method according to claim 13, wherein the prepara-
tion for cleaning radioactive liquids and surfaces is filled into
water-permeable cartridges in portions.

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US 2023/0143790 Al
12

17. The method according to claim 13, wherein the prepara-
tion for cleaning radioactive liquids and surfaces is dried by
means of lyophilisation.

18. A method for cleaning radioactive liquids, comprising
the steps of:

providing radioactive liquid to be cleaned in a reactor,

adding a preparation for cleaning radioactive liquids into

the reactor, the preparation produced by a method for
the production of a perparation for cleaning radioactive
liquids and surfaces comprising

providing ofa substance with aerobic microorganisms, par-

ticularly cultivated comprising the steps of:

providing an aqueous solution having a pH value of 5.5 to
9.0 in a container

adding a substrate in a first substrate dosage to the aqu-
eous solution,

adding further elements to the aqueous solution,

adding an inoculant with microorganisms to the aqueous
solution,

wherein an initial product is provided by the abovemen-

tioned steps, and wherein, subsequently, the following

steps are performed:

introducing air into the initial product or into intermedi-
ate products developing therefrom,

varying a termperature of the initial product or the inter-
mediate products developing therefrom in a range
from 15 to 35 degrees Celsuis,

wherein, afterhaving added the substratae in the first sub-
strate dosage, the following steps are performed for
monitoring the cultivation:

taking a reference liquid sample and determining a first
concentration of organic substance in the reference
liquid sample, substance in the reference liquid
sample,

taking another liquid sample and determining another
concentration of organic substance in the further liquid
sample after the expiration of the first waiting time,

ifthe further concentration of organic substance is smal-
lerthan 10 percent of the first concentration of organic
susbtance, adding substrate in another substrate
dosage,

repeating the abovementioned steps for monitoring the
cultivation until a sufficient amount of biomass is pre-
sent in the container, or

providing of a substance with anaerobic microorgan-
isms, particularly cultivated the anaerobic cultivation.
of microorganisms comprising the steps of:

providing an aqueous solution having a pH value of 4.5 to
7.5 ina container,

adding a substrate in a first substrate dosage to the aqu-
eous solution,

adding further elements to the aqueous solution,

adding an inoculant with microorganisms to the aqueous
solution, wherein an initial product is provided by the

May 11, 2023

abovementioned steps, and wherein, susbsequently,
the following steps are performed:

hermetically sealing the container,

varying a temperature of the initial product or the inter-
mediate products developing therefrom in a range
from 40 to 80° C.,

wherein, after having added the substrate in the first sub-

strate dosage, the following steps are performed for mon-

itoring the cultivation:

taking a reference liquid sample and determining a first
concentration of organic substance in the reference
liquid sample,

taking another liquid sample and determining another
concentration of organic substance in the further liquid
sample after the expiration of the first waiting time,

ifthe further concentration of organic substance is smal-
ler than 10 percent of the first concentration of organic
substance, adding substrate in another substrate
dosage,

repeating the abovementioned steps for monitoring the
cultivation until a sufficient amount of biomass is pre-
sent in the container.

wherein the substance is subjected to the following steps for

washing:

centrifuging the substance for producing wet biomass.

adding an isotonic solution to the wet biomass for produ-
cing a washed substance, wherein the steps for wash-
ing are repeated a plurality of times, if required, to
thereby provide the preparation for cleaning radioac-
tive liquids and surfaces,

adding a substrate into the reactor,

adding elements selected from the group of K, Ca, P, S,N,
Zn, Co, Mn, Cl, Cu, Mo, Ni, Se, B, Fe,

wherein, by the abovementioned steps, a working med-
ium is provided, and wherein, further, the following
step is performed:

removing cleaned radioactive liquid from the reactor.

19. The method according to claim 18, wherein a selection
from the group of K, Ca, P, S, N, Zn, Co, Mn, Cl, Cu, Mo, Ni,
Se, B, Fe of added elements is depleted of one or more ele-
ments which are to be understood to be biochemical equiva-
lents of the elements on which the radioactivity of the radio-
active liquid is based.

20. The method according to claim 18, wherein one or more
liquid samples are taken from the reactor and tested for their
radioactivity, and in that, when amaximum value of the radio-
activity is fallen below, the step of removing cleaned radio-
active liquid from the reactor is performed.

21. The method according to claim 18, wherein the step of
removing cleaned radioactive liquid from the reactor is per-
formed after the expiration of a predetermined time.

22-32. (canceled)

ee ee