Conversion of inorganic ions to metal sulfides by microorganisms

This invention relates to a process for reducing the concentration of heavy metal ions in an aqueous waste solution, comprising the steps of: PA0 (a) contacting the aqueous waste solution with a mixed culture of Citrobacter freundii and a dissimulatory sulfate reducer, in the presence of nutrient sufficient to satisfy the nutritional requirements of the mixed culture, for a time sufficient to produce sulfide ions from a sulfide-ion precursor in the aqueous waste solution or nutrient and to precipitate the heavy metal ions in the form of corresponding sulfides and PA0 (b) removing the thus-precipitated sulfides from the waste solution.

TECHNICAL FIELD 
This invention relates to the use of a combination of Citrobacter freundii 
and a dissimulatory sulfate-reducing microorganism to reduce sulfide-ion 
precursors to sulfide ions and bring about precipitation of heavy metal 
ions in aqueous waste solutions as corresponding sulfides. 
BACKGROUND ART 
Trace elements are widely used industrially and for medical purposes. 
Platinum, mercury, cadmium, copper, chromium and zinc are used in the 
plating industries. Lead is used to make electrodes for batteries and 
lamps. Compounds of these metals are used as catalysts for making varnish 
and paint compositions. 
Waste waters from mining and public utilities contain dissolved or 
entrained heavy metal ions. Radionuclides, particularly uranium, plutonium 
and cesium ions, are representative of objectionable and toxic components 
of waste waters from nuclear power plants or radioactive waste processing 
installations. 
Disposal of wastes from these industries presents ecological problems, 
particularly when the heavy metals are in soluble forms. The soluble forms 
are objectionable because consumption of water, containing soluble species 
of heavy metals, by humans or domestic animals can result in absorption 
and accumulation of relatively large amounts of toxic heavy metal 
compounds in the organs of animals, consuming the contaminated waters. The 
heavy metal compounds can accumulate in nervous tissue, for example, and 
cause disorders of the nervous system. There is therefore considerable 
interest in processes for removing toxic or objectionable heavy metal 
species from waste waters. 
Hallberg, in U.S. Pat. No. 4,354,937, has proposed precipitating heavy 
metals from waste waters by treating the waters with sulfate-reducing 
bacteria, particularly Desulfovibrio and Desulfotomaculum. Sulfides of 
metal contaminants in the water are precipitated and can thereafter be 
removed from the water. 
Kauffman et al. (U.S. Pat. No. 4,519,912) have recited removing both 
sulfate and heavy metals from aqueous solutions by treatment with at least 
one anaerobic Clostridium organism and a second anaerobic organism, 
selected from among genera of Desulfovibrio and Desulfotomaculum. The 
waste water is contacted with the mixture of organisms under anaerobic 
conditions. Kauffman et al. (U.S. Pat. No. 4,522,723) have proposed a 
similar process, using Desulfovibrio or Desulfotomaculum organisms, in a 
porous matrix under anaerobic conditions. 
Stern et al. (U.S. Pat. No. 4,584,271) have recited reducing sulfate to 
sulfide in a two-step bacterially-induced process, involving Desulfovibrio 
or Desulfotomaculum, under anaerobic conditions. 
Balmat (U.S. Pat. No. 4,200,523) recites removal of sulfate from aqueous 
streams by treatment with sulfate-reducing bacteria, particularly 
Desulfovibrio desulfuricans, under anaerobic conditions. 
Yen et al., in U.S. Pat. No. 4,124,501, have disclosed purifying oil shale 
retort water, using Desulfovibrio organisms to reduce sulfate to sulfide. 
The process contemplates aerobic oxidation of sulfide to sulfate, using 
aerobic organisms, such as those of the Thiobacillus family. 
Baldwin et al. (U.S. Pat. No. 4,519,913) have recited removal of selenium 
compounds from aqueous solution by anaerobic treatment in the presence of 
bacteria of the genus Clostridium. 
The metabolism of inorganic sulfur compounds by various microorganisms has 
been the subject of a review article by Peck, "Symposium on Metabolism of 
Inorganic Compounds, V. Comparative Metabolism of Inorganic Sulfur 
Compounds in Microorganisms," Bact. Rev., vol. 26 (1962), pages 67-94. 
Dean et al. (U.S. Pat. No. 3,674,428) and Fender et al. (U.S. Pat. No. 
4,422,943) are representative of references, teaching precipitation of 
heavy metal ions in waste waters by addition of an inorganic sulfide 
compound. 
It is apparent, from the amount of activity in the field, that removal of 
objectionable or toxic ionic species of heavy metals from waste waters is 
of continuing importance in providing water supplies, from recycled waste 
waters, which are safe for drinking or industrial uses. 
It is an object of this invention to provide processes by which toxic heavy 
metal species can be removed from aqueous waste solutions. 
DISCLOSURE OF INVENTION 
This invention relates to a process for reducing the concentration of heavy 
metal ions in aqueous waste solutions by the steps of: 
(a) contacting the aqueous waste solution with a mixed culture of 
Citrobacter freundii and a dissimulatory sulfate reducer, in the presence 
of an amount of nutrient sufficient to satisfy the nutritional 
requirements of the mixed culture, for a time sufficient to produce 
sulfide ions from sulfide-ion precursors in the aqueous waste solution or 
nutrient and to precipitate the heavy metal ions in the form of 
corresponding sulfides and 
(b) removing the thus-precipitated sulfides from the waste solution. 
In another aspect, this invention relates to a process for removing heavy 
metal, sulfur dioxide or sulfur trioxide contaminants from gases, 
comprising the steps of: 
(a) passing the stack gases into a mixed culture of Citrobacter freundii 
and a dissimulatory sulfate reducer, in the presence of nutrients 
sufficient to satisfy the nutritional requirements of the mixed culture; 
(b) contacting the thus-produced mixture of culture and dissolved or 
entrained stack gas components for a time sufficient to convert sulfur 
dioxide, sulfur trioxide or other sulfide precursors in the stack gases or 
nutrient to sulfide; 
(c) precipitating heavy metals as corresponding sulfides; and 
(d) removing the thus-precipitated metal sulfides from the mixture of 
culture and dissolved stack gas components. 
In a further aspect, this invention relates to a method for immobilizing 
heavy metal ions in soil samples, containing sulfate-reducing 
microorganisms, comprising adding to the soil sample sufficient 
sulfide-precursor compound to cause conversion of the sulfide precursor to 
sulfide and to precipitate heavy metal ions as corresponding sulfides. 
For the practice of this invention, a culture of a mixture of Citrobacter 
freundii and a dissimulatory sulfate reducer has been deposited with the 
American Type Culture Collection, Bethesda, Md., and is designated ATCC 
53512. 
In the event that during the pendency of this application, the Commissioner 
of Patents and Trademarks shall determine that some individual is entitled 
to receive progeny of this strain in accordance with the provisions of 37 
C.F.R. 1.114 and 35 U.S.C. 122, the required written authorization will be 
provided by the assignee of this application. 
Upon the issuance of this application as a patent, a culture of this 
mixture can be obtained from the permanent collection of the American Type 
Culture Collection. 
Among dissimulatory sulfate reducers, useful in the practice of this 
invention, are organisms of the genus Desulfomonas. Particularly preferred 
are organisms of the species Desulfomonas pigna and related subspecies. 
Although it will be understood that any of the Desulfomonas species, 
particulary Desulfomonas pigna, can be used under anaerobic conditions to 
reduce sulfide-precursors to sulfide, it is preferred to carry out the 
process under aerobic conditions, using in combination with Desulfomonas a 
culture of Citrobacter freundii, which provides the necessary conditions 
for reducing precursors to sulfides. 
The mixed culture of Desulfomonas and Citrobacter freundii requires a 
minimum medium containing yeast extract, ascorbic acid or cysteine, sodium 
acetate or lactate, magnesium sulfate, dipotassium hydrogen phosphate, 
ammonium sulfate and sodium chloride. The pH of the medium is adjusted to 
5-9, preferably to 6-8 with an acid, such as hydrochloric acid. 
The minimal medium preferably contains, per liter: 
______________________________________ 
grams 
______________________________________ 
1-15 yeast extract 
0.1-1.5 ascorbic acid or cysteine 
0.05-0.3 sodium lactate or sodium acetate 
0.5-4 magnesium sulfate 
0.05-0.3 dipotassium hydrogen phosphate 
0.05-0.3 ammonium sulfate 
0.1-1.5 sodium chloride 
______________________________________ 
The foregoing medium is exemplary of nutrient "sufficient to satisfy the 
nutritional requirements of the mixed culture," as used in the 
specification and claims. 
Contemplated equivalents of the minimum medium, providing support for the 
nutritional requirements of the mixed culture of Desulfomonas and 
Citrobacter freundii, include, but are not limited to, media containing: 
(a) sodium lactate or sodium acetate 
(b) mineral salts 
(c) sulfate 
(d) carbon source, e.g., yeast extract and 
(e) ascorbic acid or cysteine. 
Sulfide precursors include, but are not limited to, sulfate, sulfite, 
thiosulfate, organic sulfates and similar organic or inorganic species. 
The sulfide precursors can be present in the aqueous waste solutions, for 
example, those containing dissolved stack gases from power plants. Removal 
of at least some of the sulfide precursors, particularly dissolved sulfur 
dioxide and sulfur trioxide, in inherent to the process. If the waste 
solutions contain insufficient sulfide precursors to precipitate all of 
the heavy metal ions dissolved therein, sulfide precursors are normally 
added in the form of sulfate ions. 
The aqueous waste solution is contacted with the mixed culture of 
Desulfomonas and Citrobacter freundii at temperatures ranging from about 
5.degree. to about 35.degree. C. However, for optimum culture growth, it 
is preferred to contact the waste waters with Desulfomonas and Citrobacter 
freundii cultures at higher temperatures, more preferably from 15.degree. 
to about 35.degree. C. 
The process of this invention is used to precipitate significant amounts of 
heavy metal compounds from waste waters and is used for the reducing the 
concentration of metal compounds at levels as high as about 50 ppm. 
However, it is preferred to operate the process using feeds containing 
from a few ppb to about 25 ppm of ionic forms of heavy metals. 
Feeds which are preferably processed in accordance with the invention 
include (a) aqueous waste solutions from metal plating, paint 
manufacturing or mining effluents; (b) waste solutions containing 
radionuclides from nuclear power plants or radioactive waste processing 
and (c) scrubbing solutions for absorption of heavy metal wastes from 
stack gases. 
The time for contacting the aqueous waste solutions with Desulfomonas and 
Citrobacter freundii is selected so as to bring about essentially complete 
precipitation of heavy metal compounds as their sulfides. The time will in 
part be a function of the amount of sulfide-precursors in the waste waters 
or the nutrient medium. The time required can be as little as 5-10 minutes 
when the aqueous waste is contacted with confluent cells. Longer treatment 
times, of the order of 12 hours or more, can be required when growing 
mixed cultures are used. It will be understood that those skilled in the 
art can determine preferred and optimum contact times by routine 
experimentation. 
The heavy metal ions, removed in accordance with the process of this 
invention, include, but are not limited to Cd.sup.++, Hg.sup.++, 
Ni.sup.++, Zn.sup.++, Cu.sup.++, Fe.sup.++ and Pb.sup.++. Treatment of 
wastes, containing at least 1 ppm of one or more of these metals, done in 
a continuous mode using at least 10.sup..ident. cells/mL of Desulfomonas 
and at least 10.sup.2 cells/mL of Citrobacter freundii, normally requires 
contact times of 12-48 h. Batch treatment of such feeds, using at least 
10.sup.8 cells/mL of Desulfomonas and at least 10.sup.3 cells/mL of 
Citrobacter freundii, requires contact times of 3-12 h. 
Waste waters are preferably contacted with the mixed culture under aerobic 
conditions. The process of the present invention is therefore preferably 
carried out under ambient conditions, without exclusion of air from the 
container in which the treatment is being done. Most preferably, nitrogen 
is bubbled through the mixture of waste water and treatment during the 
process. A preferred flow rate is 0.6 mL/min. 
Precipitation of sulfides of the heavy metals can occur outside the cells 
of the mixture, in cells, or on the surface of the cells. In any case, the 
insoluble sulfides can be removed from the waste waters being treated by 
filtration through a filter of suitable pore size. 
The removal of heavy metals, in the form of sulfides, from representative 
contaminated water streams was demonstrated by atomic absorption 
spectroscopy of material collected on filters, and dissolved in nitric 
acid, after specified incubation times. The removal of heavy metal ions 
from the solutions being contacted was almost quantitative, provided the 
contact time was sufficiently long. 
The waste solutions can be contacted with the mixed culture in an 
essentially aqueous medium, for example, by adding culture and nutrients 
to the waste solutions in the form of a broth. At the end of the contact 
time, the incubate can be filtered to remove insoluble materials and the 
filtrate, which is essentially free of heavy metal contaminants, can be 
recycled to a chemical process or discharged into a stream. The residue on 
the filter, containing metallic sulfides, can be treated in conventional 
ways to recover the metals. 
Alternatively, the waste waters can be contacted with supported cultures of 
the Desulfomonas and Citrobacter freundii organisms. The waste waters can 
be passed through one or more beds of microorganisms, immobilized on, or 
adhered to beads of plastic, alginic acid or the like. It is preferred to 
use a bed of microorganisms, supported on alginic acid, because the 
process can be carried out continuously and the beds can be withdrawn from 
service periodically to recover heavy metals sulfides therefrom. When the 
process is being done using supported microorganisms, it will be 
understood materials required for satisfying the nutritional requirements 
of the microorganisms of the mixed culture are added to the aqueous waste 
feed. The pH of the feed stream will also be adjusted, as necessary. 
The process of this invention can be carried out in several stages. When 
the aqeuous feed contains high concentrations of heavy metal compounds, it 
is preferred to treat the feeds in a series of treatment steps, each of 
which results in reducing the concentration of heavy metal ions in the 
feed stream. 
BEST MODE FOR CARRYING OUT THE INVENTION 
In a most preferred embodiment, the process of this invention is carried 
out at pH 6-8, at temperatures of 15.degree.-35.degree. C., under aerobic 
or anaerobic conditions in the presence of a mixed culture of Desulfomonas 
pigna and Citrobacter freundii, having the identifying characteristics of 
ATCC 53512.

Without further elaboration, it is believed that one skilled in the art 
can, using the preceding description, utilize the present invention to its 
fullest extent. The following preferred specific embodiments are, 
therefore, to be construed as merely illustrative and not limitative of 
the remainder of the disclosure in any way whatsoever. 
In the following examples, temperatures are set forth uncorrected in 
degrees Celsius. Unless otherwise indicated, all parts and percentages are 
by weight. 
EXAMPLE 1 
Minimal Medium for Desulfomonas and Citrobacter freundii 
Minimal medium concentrate is prepared by dissolving ingredients, shown in 
Table 1, in 1000 mL of distilled water. The pH of the solution is adjusted 
to 7.5 using hydrochloric acid. The concentrate is sterilized by 
autoclaving. 
Medium is prepared by diluting 100 mL of concentrate with 900 mL of 
distilled water. 
EXAMPLE 2 
Precipitation of Heavy Metal Ions as Sulfides by Action of a Mixed Culture 
of Desulfomonas and Citrobacter freundii 
A test culture containing 10.sup.8 cells/mL of Desulfomonas and 10.sup.5 
cells/mL of Citrobacter freundii (ATCC 53512) is made using minimal medium 
of Example 1, to which is added trace amounts of heavy metal cations. The 
cultures are incubated at 32.degree. C. under aerobic conditions with 
shaking for the indicated time. Aliquots (3 mL) are removed and filtered 
on a membrane filter (0.45 micron filter), which is digested in nitric 
acid. Atomic absorption spectroscopic measurements of the nitric acid 
solution permit determination of the amount of trace element on the 
filter. Results are shown in Table 2 and represent the mean.+-.SD for sets 
of three experiments. These results show that significant reduction of 
heavy metal ion concentration occurs within 6 hr and that heavy metal ions 
are almost completely removed within 48 h. 
Controls are culture-free incubates, which are sampled at the same 
intervals as the test cultures. Heavy metal removal for the controls is 
10-15% of the heavy metal, initially present. 
TABLE 1 
______________________________________ 
Minimal Medium Concentrate for Sulfate Reducing Bacteria.sup.(a) 
grams/1000 mL solution.sup.(b) 
______________________________________ 
Yeast extract 100 
Ascorbic acid or cysteine 
10 
Sodium lactate or sodium acetate 
1 
MgSO.sub.4 20 
K.sub.2 HPO.sub.4 1.0 
(NH.sub.4).sub.2 SO.sub.4 
1.0 
NaCl 100 
______________________________________ 
.sup.(a) The broth was prepared by diluting 100 mL of the concentrate wit 
900 mL of distilled water. 
.sup.(b) The solutes were brought to a volume of 1000 mL with distilled 
water and pH adjusted to 7.5. The concentrate was sterilized by 
autoclaving before use. 
TABLE 2 
______________________________________ 
Metal Sulfide Formation as a Function of Incubation Time for 
Solutions of Heavy Metal Ions with Citrobacter freundii and 
Desulfomonas.sup.(a) 
Concentration 
of inorganic 
Incubation Time (h) 
element added 
0 6 12 24 48 
(ppm) % recovery in the filtrate 
______________________________________ 
1 CaCl.sub.2 
95 .+-. 6 75 .+-. 
21 .+-. 8 
10 .+-. 3 
12 .+-. 6 
8 
7 HgCl.sub.2 
90 .+-. 12 
50 .+-. 
15 .+-. 6 
6 .+-. 1 
5 .+-. 3 
8 
10 NiCl.sub.2 
88 .+-. 10 
77 .+-. 
20 .+-. 8 
17 .+-. 8 
10 .+-. 4 
10 
1 ZnCl.sub.2 
93 .+-. 9 51 .+-. 
10 .+-. 2 
7 .+-. 6 
9 .+-. 6 
7 
5 CuCl.sub.2 
100 .+-. 2 
63 .+-. 
40 .+-. 6 
30 .+-. 10 
28 .+-. 9 
12 
10 FeCl.sub.2 
95 .+-. 8 
79 .+-. 
25 .+-. 3 
18 .+-. 3 
5 .+-. 6 
9 
10 PbCl.sub.2 
91 .+-. 5 79 .+-. 
12 .+-. 8 
8 .+-. 3 
3 .+-. 2 
8 
______________________________________ 
.sup.(a) Mean .+-. SD for 3 experiments. 
EXAMPLE 3 
Comparison of Batch and Continuous Treatment of Solutions of Heavy Metal 
Ions 
Batch experiments are carried out by contacting medium of Example 1, 
containing 5 ppm of an indicated heavy metal chloride, with 10.sup.12 
cells/mL of Desulfomonas and 10.sup.5 cells/mL of Citrobacter freundii. 
The cultures are incubated at 32.degree. C. for 5 h, after which 10-mL 
aliquots of the cultures are filtered through a membrane filter as in 
Example 2. Heavy metal ion concentration is measured by atomic absorption 
spectroscopy. 
Continuous contacting of minimal medium, containing 5 ppm of an indicated 
metal chloride, is carried out using culture containing 10.sup.9 cells/mL 
of Desulfomonas and 10.sup.5 cells/mL of Citrobacter freundii. Incubation 
is continued for 24 h at 32.degree. C. Aliquots are removed and analyzed 
as above. Results are shown in Table 3. These results shows that almost 
complete removal of objectionable metal ions can be accomplished by either 
batch or continuous processes. 
EXAMPLE 4 
Effect of Sulfate Addition on Activity of Soil Samples 
Soil samples are treated with a culture of Desulfomonas (10.sup.8 cells/mL) 
and Citrobacter freundii (10.sup.4 cells/mL) at 30.degree. C., with and 
without added sulfate. Sulfate, if added, is a 1:1 mixture by weight of 
ferric ammonium sulfate and magnesium sulfate. Sulfate-reducing activity 
is indicated by formation of black precipitate in the soil sample within 
24 h after mixing the cultures with soil samples. 
Results are shown in Table 4. These samples show that addition of 100 ppm 
of sulfate greatly improves sulfate-reducing activity of the mixed culture 
and that sulfate levels of 200 ppm permit reduction in all cases. 
EXAMPLE 5 
Effect of Adding Calcium Sulfate to Soil Columns on Leaching of Mercury 
A solution of minimal medium, containing 1000 ppb of mercury (as mercuric 
chloride), is treated with Desulfomonas and Citrobacter freundii (10.sup.8 
and 10.sup.5 cells/mL, respectively) at 30.degree. C. Calcium sulfate (500 
ppm with respect to soil) is added at the top of the column or mixed with 
the soil. The amount of mercury leached out of the soil column by passage 
of deionized water through the column at a rate of 1 mL/h is determined by 
atomic absorption spectroscopy (cold vapor technique). 
As shown by the results in Table 5, addition of a sulfate to a soil sample 
markedly reduces the rate at which mercury is leached from the sample. 
TABLE 3 
__________________________________________________________________________ 
Comparison of Batch and Continuous Processes for Sulfidation of Heavy 
Metal Ions 
% Recovery of Metal Added.sup.(c) 
Process 
Conditions 
Fe Cd Zn Ca Hg Pb 
__________________________________________________________________________ 
Batch Control 10 .+-. 6 
9 .+-. 3 
14 .+-. 8 
18 .+-. 7 
6 .+-. 6 
5 .+-. 2 
Experimental.sup.(a) 
93 .+-. 6 
88 .+-. 8 
90 .+-. 10 
89 .+-. 3 
94 .+-. 8 
90 .+-. 12 
Continuous 
Control 12 .+-. 8 
14 .+-. 4 
10 .+-. 2 
20 .+-. 3 
11 .+-. 6 
8 .+-. 2 
Experimental.sup.(b) 
84 .+-. 15 
93 .+-. 8 
80 .+-. 12 
77 .+-. 5 
91 .+-. 18 
99 .+-. 7 
__________________________________________________________________________ 
.sup.(a) Cells added at concentrations of 10.sup.12 cells/mL for 
Desulfomonas and 10.sup.5 cells/mL for Citrobacter freundii; incubation 
period 5 h. 
.sup.(b) Cells added at concentrations of 10.sup.9 cells/mL for 
Desulfomanas and 10.sup.5 cells/mL for Citrobacter freundii; incubation 
period 24 h. 
.sup.(c) Results are mean .+-. SD for 3 experiments. 
TABLE 4 
______________________________________ 
Sulfate-reducing Activity in Amended Soil Samples 
as a Function of Sulfate Concentration.sup.(a) 
Sulfate added to soil (ppm) 
Sample No. 0 100 200 
______________________________________ 
1 - + + 
2 - + + 
3 - + + 
4 - + + 
5 - + + 
6 - - + 
7 - + + 
8 - + + 
9 - - + 
10 - + + 
11 - - + 
______________________________________ 
.sup.(a) Sulfatereducing activity is indicated by + and inactivity by 
TABLE 5 
______________________________________ 
Effect of Adding CaSO.sub.4 on Leaching of 
Mercury from Soil Columns 
Time (days) 
7 42 84 
Hg Leaching (ppb) 
______________________________________ 
Controls 3.9 2.34 0.69 
CaSO.sub.4 (500 ppm) 
0.88 0.20 0.23 
added to top of 
column 
CaSO.sub.4 (500 ppm) 
1.3 0.35 0.23 
mixed with soil 
______________________________________ 
EXAMPLE 6 
Determination of Optimum Conditions for Reduction of Sulfide-ion Precursors 
to Sulfide Ions 
Minimal medium, as in Example 1, containing yeast extract, ascorbic acid or 
cysteine, sodium lactate or acetate, magnesium and ammonium sulfates, 
dipotassium hydrogen phosphate and sodium chloride, is preferred. 
For a continuous process, preferred concentrations of Desulfomonas are 
10.sup.6 -10.sup.10 cells/mL and of Citrobacter freundii, 10.sup.3 
-10.sup.7 cells/mL. 
For batch processes, cellular concentrations of 10.sup.9 -10.sup.13 
cells/mL of Desulfomonas and 10.sup.4 -10.sup.8 cells/mL of Citrobacter 
freundii, are preferred. 
Although conversion of sulfate to sulfide is a reductive process, the 
process can be carried out under aerobic conditions using Citrobacter 
freundii in combination with Desulfomonas. 
The preceding examples can be repeated with similar success by substituting 
the generically or specifically described reactants and/or operating 
conditions of this invention for those used in the preceding examples. 
From the foregoing description, one skilled in the art can easily ascertain 
the essential characteristics of this invention and, without departing 
from the spirit and scope thereof, can make various changes and 
modifications of this invention to adapt it to various usages and 
conditions.