OCCURRENCE OF MERCURY-RESISTANT MICROORGANISMS IN
MERCURY-CONTAMINATED SOILS AND SEDIMENTS IN
PAVLODAR, KAZAKHSTAN
Svetlana A. Abdrashitova (Institute of Microbiology and Virology, Ministry of
Education and Science, Kazakhstan)
Rathi G. Kavanaugh (University of Cincinnati, Cincinnati, Ohio)
M.A. Ilyushchenko (Al-Farabi Kazakh State National University, Kazakhstan)
A.Yu. Kalmykv, and S. A . Aitkeldieva (Institute of Microbiology and Virology,
Ministry of Education and Science, Kazakhstan)
Brian J. Morris (University of Cincinnati, Cincinnati, Ohio)
Richard D ever eux (U.S. EPA, Gulf Breeze, Florida)
Wendy J. Davis-Hoover (davis-hoover.wendy@epa.gov) (U.S. EPA, Cincinnati, Ohio)
ABSTRACT: There is extensive mercury contamination of soil surrounding a
chloralkali plant in Pavlodar, Kazakhstan, that operated from 1970 to 1990. High-level
mercury contamination exists within the confines of the plant, at nearby off-site waste
storage and evaporation ponds, and in Balkyldak Lake, which adjoins the waste ponds.
The Irtysh River, a major river in Kazakhstan and a drinking water source, is threatened
by a plume of mercury-contaminated groundwater and may already be affected.
Estimates of the extent and levels of contamination were made based on available
information, the amount of mercury used at the plant over the period it operated and field
monitoring. Bacteria, Actinomyces sp., and fungi from soil and sediment samples
collected at the plant and Balkyldak Lake were cultured. A high percentage of these
microorganisms were resistant to mercury chloride concentrations of up to 0.1 mM.
These mercury-resistant microorganisms are being characterized for their potential to be
used in bioremediation at this site and other contaminated sites in Kazakhstan and the
world.
INTRODUCTION
Mercury is a naturally occurring element in the earth's crust. Anthropogenic
emissions and releases account for some of the largest inputs of mercury to the
environment. Exposure of animals and humans to mercury can cause numerous ill health
effects. Historically, large quantities of mercury have been used in the extraction of gold,
in chloralkali plants, as pesticides and in the medical and dental fields. Significant
release of mercury can occur at chloralkali plants used in the production of chlorine.
Chlorine is produced by the amalgam process involving electrolytic dissociation of
chloride solutions with Hg° serving as the cathode. Production of chlorine in this manner
accounts for 60% of the total production in Europe.
Some areas in republics of the former Soviet Union are heavily contaminated with
mercury as a result of releases from industrial plants. These include industrial zones in
the following cities: (1) Sumgait in Caucasus, (2) Volgograd, Belgorod, Shvarts, Kirovo-
Chepetsk, Sterlitamak in the European part of Russia, (3) Usolie-Sibirskoe and Sayansk
in Siberia, and (4) Temirtau and Pavlodar in Kazakhstan. Ingress of mercury from the
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plants to the environment has resulted in significant pollution of soils, silts ground and
surface waters. Although pollution of groundwater and surface water by other toxic
metals, such as arsenic, copper, and lead is more common than mercury pollution, the
high levels of mercury encountered impose a considerable risk to the populations of these
regions.
The northern suburb of Pavlodar City is contaminated by mercury as a result of
activity at a chlor-alkali chemical plant (Khimprom), which produced chlorine from the
1970s to the 1990s. It is estimated that 1000 tons (10 tons of which is HgCb) of mercury
lay in the soil 2-4 m beneath the plant. Soil and groundwater in the surrounding vicinity
of the plant are also contaminated with high concentrations of mercury. The plant is 5km
from the Irtysh River, a major river in Kazakhstan. According to some reports, a plume
of mercury from the plant has already reached the river. In addition, an estimated 10 tons
of mercury in wastewater was discharged into Balkyldak Lake during operation of the
plant.
To date, the amount of mercury released from chlor-alkali plants in Kazakhstan
has not been recorded or published. Therefore, we will consider estimates of the total
maximum amount of mercury possibly released by the plant in Pavlodar to provide
insight into the potential magnitude of the problem. The total amount of mercury used at
Pavlodar in the electrolytic process at any time was 187 tons. If this entire amount were
released each year the total amount released for the entire time the plant was in operation
(19 yrs) would be 3550 tons. We know however, that some amount of mercury was
recycled as part of the process. Another estimation can be based on the consumption of
mercury by the plant. Annual mercury consumption was estimated to be 120 tons
(Novosibirk. 1995). If 75% of this is assumed to be lost every year, then about 1700 tons
was released during operation of the plant. The actual total losses of mercury are maybe
closer to 3000 tons and are quite likely more than 1700 tons.
Estimates can also be based on the averaged concentrations of mercury measured
in the plant's buildings, structures, soils, wastes and adjoining sediments of Balkyldak
Lake. Other factors and any longer distance dispersion of mercury are not included in the
estimation. Figure 1 is a map of the chloralkali plant and its vicinity showing the extent
of mercury contamination. Studies carried out within the confines of the chemical plant
showed it is intensively contaminated with mercury (Sintez, 1991; KffiF
GOSNIMLORPROEKT, 1989a; NTS Tehnolog, 1990; HIF GOSNIMLORPROEKT,
1989b; KNII Sinteko, 1992.). The total aerial extent of contaminated soil at the plant is
521150 m . Soils in the area east of the electrolysis workshop are the most contaminated.
There, the average concentration of mercury in topsoil (0-25cm) is 14 mg/kg. High
contaminations of mercury were found more than 4 m below the surface and mercury
concentrations exceeded the maximum permissible concentration (MPC) to depths of
approximately of 1.5 m. From these data, release of mercury from the plant can be
considered to have been about 1400 tons minimally with a maximum amount of
approximately 3500 tons.
Spills of process saline solutions and water around the electrolysis workshop
building resulted in centers of highly saline groundwater containing chlorides and sodium
up to 62-72 g/1 and extremely high concentrations of mercury (12.5-103.0 mg/1; 25000-
206000 times higher of MPC which is 0.5 ju.g/1 for water). Mercury has spread in the
southwest, western, northwest and northern directions up to 800 m away from the
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workshop. The aerial extent of the contaminated first horizons of groundwater and
stationery groundwater is 0.55 km2; the total content of mercury dissolved in them is
about of 10 tons.
Balkyldak Lake had once been used to receive considerable volumes of mercury-
containing wastewater from the Khimprom plant. This has resulted in the accumulation
of high concentrations of mercury in bottom sediments (up to 1200 mg/kg). The
concentration of mercury in the lake water reached 25 mg/1 (50000 times higher than the
MPC) during some periods. Subsequently, evaporation storage ponds were constructed
to receive waste from the plant. Mercury in one of the evaporation ponds receiving
wastes from the plant reached 2.3-51.5 mg/1 (4600-10300 times higher of MPC) and the
accumulated solid material enriched with mercury is estimated at 270,000 tons. After the
wastewater storage ponds had been put into operation, the mercury levels in the water of
Balkyldak Lake decreased and varied in the range of 0.001-0.01 mg/1 (2-20 times higher
of MPC). However, the ponds were constructed adjacent to the lake and, because of
periodic or emergency discharges of wastewater from the ponds, mercury levels in the
lake could sharply rise. For example, in 1992 mercury concentrations in the lake water
increased to 10 mg/1.
High concentrations of mercury at the waste ponds resulted in a gradual extension
of contaminated groundwater (more then MPC, i.e. 0.5 |ig/l) in the area. In 1985,
mercury above the MPC was discovered only in wells located 10-150 m to the south and
southwest of the bank line, and the area of the contaminated ground water was 2-3 km .
In 1987, the contaminated area increased up to 12-15 km2 in the same direction. In
addition, centers of groundwater contamination (up to 0.4-0.5 km ) were noted in
northern and eastern directions.
Mercury concentrations found in snow exceeded the MPC of 0.5 |ig/l over an area
of about 255 km2. Mechanisms of mercury migration were probably wind transport of
dust, snow and ice from the surface of Balkyldak Lake and the wastewater storage ponds,
and dispersion of ventilation releases from the electrolysis workshop at Khimprom plant.
In 1992, a considerable decrease in the area of contaminated snow cover was observed
when there was a reduction in the industrial activity of the chemical plant. This further
supports the likelihood of dust and gas emissions from the plant as a major source of the
mercury transported to the atmosphere and deposited on the ground.
Integrated studies of mercury distributions in (1) the water and sediments of
Balkyldak Lake, (2) in groundwater in the vicinity of the plant, (3) in snow cover, (4) in
the waters and sediments of the waste and storage ponds, including an ash lagoon, and (5)
nearby reservoirs in the northern industrial section of Pavlodar City has confirmed a high
level of industrial contamination in this region (Zhetekshi 1988,1991,1993)
At present various projects have been proposed to mitigate the contamination
under and around the electrolysis workshop since these are the areas with the highest
mercury levels. These areas are a source for mercury input by groundwater ingress into
the waters of Balkyldak Lake and Irtysh River. However, even after the implementation
of any demercurization project for the Khimprom plant, a substantial amount of mercury
will still remain in the topsoil and beneath the surface at the plant and waste ponds. This
mercury will continue to be a source of pollution entering the Balkyldak Lake and Irtysh
River via groundwater inflow.
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Research into further determining the extent of contaminated soil and sediments
around the plant, and a study into the microbial communities at contaminated areas is
underway by an international team of scientists from Kazakhstan and the U.S.A. Soil and
sediment samples were obtained from contaminated sites around the electrolysis plant.
Sample sites and sampling methodology are described in a section below. The samples
were analysed by researchers in Khazakhstan and in the U.S.A. These research efforts
are aimed at isolating mercury resistant bacteria from contaminated soils for possible
future bioremediation applications.
MATERIALS AND METHODS
Sample Collection. Samples of soil were collected from areas near the electrolysis
workshop at the Khimprom plant in Pavlodar in September 2001. Soils and sediments
were also obtained near waste storage ponds used by the Khimprom plant, and from the
edge of Balkyldak Lake. Soil samples, about 1 cm beneath the surface, were collected
using spatulas. Sediment samples were collected using a 170 mm x 60 mm diameter
coring device. Samples were placed in acid-washed and sterilized plastic bottles or
whirlpak bags.
Locations of the sampling sites (1-13) are shown in Figure 1. Samples labeled P
were refrigerated within 2 days and processed in the Almaty, Kazakhstan laboratories
within one month. Samples labeled U were shipped to the EPA laboratory in Cincinnati
and processed within one month. The holding temperature for the U samples during the
25day transit is not available, but is likely to be variable room temperatures. The U
samples were stored at 4°C upon receipt at the laboratory.
Analyses of Samples in the Kazakhstan Laboratory. Soil and silt samples were
inoculated onto plates containing the following nutritious media: fish peptone agar
(FPA), peptone iron agar (PIA), and R2A agar. To identify resistance of microorganisms
to mercury, 0.01 and 0.1 mM of HgCb were added to the media. Controls consisted of
media without added mercury. Colonies of bacteria, fungi and Actinomyces sp. were
identified by colony morphology.
Analyses of Samples in the U.S Laboratory. An initial screening for microorganisms
was done using the 'U' soil samples. One gram each of the soil samples was extracted
using 10 mL of sterile deionized (DI) water. The soil samples were added to flasks
containing 10 mL of the sterile DI water and shaken on a shaker for 2 hrs at 150 rpm.
Undiluted extracts were plated on YM agar (Glucose-10.Og; Peptone-5.0g; Yeast Extract-
0.3g; Malt Extract-0.3g) supplemented with mercuric chloride. Three different
concentrations of mercury, 0.005mM (1.36mg/l), 0.02mM (5.43mg/L) and 0.05mM
(13.6mg/L) in plates were tested.
Based on the results from the initial screening, second plating was done using YM
agar supplemented with 0.05mM HgCh in order to enumerate mercury resistant bacteria
and unsupplemented-YM and R2A agars for total counts of bacteria. Dilutions of the soil
extract were prepared in Butterfield's phosphate buffered solution (U.S. FDA). A stock
solution of KH2PO4 was prepared in DI water. 1.25 mL of the stock was diluted to 1L
and the pH was adjusted close to 7.2 and the buffer was sterilized. This buffer solution
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was used in the preparation of dilutions. Fungi and Actinomyces were identified by
colony morphology. Pure cultures of selected microbes were isolated on YM plates
containing 0.05mM HgCh. The cultures were stored at -80°C.
FIGURE 1. Focus of mercury pollution in the North Industrial Site of Pavlodar
City, KZ. Light boreholes (circles) in A and B contained mercury concentrations
greater than the MPC in groundwater samples. Black boreholes did not contain
mercury in the ground water samples. The three sludge lagoons circled in B are
together about 1 KM in length.
RESULTS AND DISCUSSION
Table 1 shows the results of analyses done at the Kazakhstan laboratory. There
was variation in the number of aerobes and saprophytic microorganisms recovered on
different media. The results also showed that the isolated microorganisms were tolerant
to 0.001 mM HgCb in the media. Increasing the HgCh concentration in the media to 0.1
mM however, resulted in a reduction in the recovery of microorganisms. Fungi and
Actinomyces were found only in the soil (not in silt samples) and found to grow in media
with HgCb contents of 0.1 mM. On PIA and some PFA agar plates however, the
numbers from soil samples increased with an increase in the HgCb concentration in the
media.
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A total of 125 bacteria strains, 1 fungus and 4 Actinomyces were isolated from
the plates containing HgCh. Cultivation of pure cultures of bacteria in liquid complex
media with HgCh has shown that only 50 strains of bacteria were resistant to 0.01 mM
HgCh and 41 strains of bacteria to 0.1 mM HgCh. Fourteen isolated strains of bacteria
were gram-negative and 27 were gram-positive. These data show that mercury
contaminated soils and silts from the chlor-alkali plant Pavlodar City contain a wide
variety of microorganisms resistant to high levels of mercury.
Results from the analysis conducted in the EPA lab are shown in Table 2.
Undiluted extracts from the initial screening (U samples) plated on mercury-
supplemented YM plates resulted in colonies too numerous to count. Fewer colonies
grew on plates with increasing concentrations of mercury. Colonies from plates
containing 0.05mM mercury were picked to isolate mercury resistant bacteria. Unique
colony types on the 0.005mM plates not seen on 0.05mM plates were also selected. Plate
counts of total soil bacteria recovered on YM agar, R2A agar and YM agar supplemented
with HgCb at a concentration of 0.05mM are shown in Table 2.
The results are represented as counts per gram of soil. R2A agar (without added
mercury) yielded a higher total count of bacteria from all the samples. Comparisons of
counts on YM agars with and without mercury (Figure 2) show that for five of the six
samples, 16-54 percent of the total soil bacteria are resistant to a mercury concentration
of 0.05mM.
Comparison of the results in Table 1 and 2, show the counts of mercury resistant
bacteria obtained in both screenings are within the same order of magnitude. The
concentrations used by researchers in Kazakhstan were O.OlmM and O.lmM while
0.05mM were used in the U.S. laboratory. The concentration of mercury also does not
seem to have a large effect on the recovery of bacteria for many of the sites. Low
numbers at O.lmM concentration were seen on R2A supplemented with mercury (Table
2). These results suggest that the very high concentrations of mercury at the sample sites
naturally selected for survival of mercury resistant microorganisms.
Pigment-producing bacteria were found in U samples from all the sites. Many
different pigment colors were observed. Approximately 132 cultures of bacteria and
Actinomycetes were isolated from the soil samples in Table 2.
The focus of the research was to isolate microorganisms capable of growth in the
presence of high concentrations of mercury that might be useful in the mitigation of soils
and sediments and ground water. Numbers of total soil bacteria and mercury resistant
bacteria are difficult to extrapolate to the sampling sites because of errors associated with
extended holding times. In addition, it is widely recognized that not all the
microorganisms inhabiting soils can be extracted or recovered on plates. The numbers
however, show a relative abundance of the resistant microorganisms obtained in culture
from site to site.
CONCLUSIONS
Operation of the chlor-alkali facility at the Khimprom chemical plant in Pavlodar
City, Kazakhstan, resulted in high-level mercury contamination of soil within the plant
and ground water flowing beneath it. It is likely that Irtysh River, one of Kazakhstan's
main rivers, will become contaminated, if not already, by a plume of mercury in
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TABLE 1. Quantity of aerobes in mercury contaminated soil and silts of
Pavlodar City (counts per lg of soil or silt) F= fungi, v4=Actinomyces
Sample
Sampling
PFA
R2A
PIA
Number
Without
0.01 mM
0.1 mM
Without
0.01 mM
0.1 mM
Without
0.01 mM
0.1 mM
location
HgCl2
HgCl2
HgCl2
HgCl2
HgCl2
HgCl2
HgCl2
HgCl2
HgCl2
PI
Soil near
the
3.8xl06
5.2xl06
4.6xl06
9x106
9xl06
7x104
8x105
3xl06
4x106
workshop
(F: 104)
(A:
5xl04)
south-west
P2
Soil near
the
5.6xl06
3.6xl06
7x106
6.7xl06
107
1.6xl05
1.8xl06
5xl06
9x106
workshop
(F: 2x104)
(A: 104)
(A:
3xl04)
south-east
P3
Soil near
104
3.6xl06
tanks
3.3xl06
3.5xl06
2.3xl06
4.2xl06
3.1xl06
(A: 104)
1.7xl05
106
(A: 104)
P8
Lake
sediment
1.7xl06
l.lxlO6
1.9xl05
2x106
2.4xl06
0
4.6xl05
3.8xl05
5.5xl05
N 3,
surface
layer
P9
Lake
sediment
1.8xl07
1.2xl07
2.6xl06
1.5xl07
3.7xl06
4x104
1.6xl07
1.2xl07
3xl06
N 3,
the depth
is of 5 cm
P10
Lake
sediment
2.2xl06
9xl05
1.6xl05
4.5xl06
2.8xl06
104
7x105
7x105
3xl05
N 3,
the depth
is of 10 cm
Pll
Lake
sediment
3.2xl06
2.6xl06
106
2.4xl06
2.8xl06
7x104
1.4xl06
1.8xl06
9x104
N 1,
surface
layer
P12
Lake
sediment
4.8xl06
3.2xl06
2x106
7x106
5xl06
0
3.4xl06
4.2xl06
2.6xl06
N 1,
the depth
is of 5 cm
TABLE 2. Average counts of microbes on media with and without added mercuric
chloride. Standard deviations are shown in parentheses.
Sample #, location
R2A (no added Hg)
(CFU/g of soil)
YM (no added Hg)
(CFU/g of soil)
YM and
0.05mM HgCl2
(CFU/g of soil)
U2, near electrolysis
workshop
2.07(±0.17)xl0v
1.30(±0.22)xl07
3.70 (±1.59)xlOb
U8, lake shore
1.22(±0.14)xl07
5.23(±2.87)xl0b
2.83(±0.35)xl0b
U12, lake shore
1.56(±0.43)xl07
7.53(±0.45)xl0b
2.60(±0.35)xl0b
U16, lake shore
2.95(±0.1)xl07
2.68(±0.25)xl07
4.30(±0.17)xl0b
U17, lake shore
8.20(±0.7)xl0b
7.20(±0.46)xl0b
1.23(±0.35)xl0b
U18, lake shore
7.77(±1.42)xlOb
4.73(±1.27)xlOb
2.67(±0.58)xl0:>
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KHTF GOSNIIHLORPROEKT. 1989b. Recommendations for demercurization of
equipment and burial of wastes of chlorine and caustic soda production on mercury
method at Pavlodar Chemical Plant. KHIF GOSNIIHLORPROEKT, 1989. P 41.
Novosibirsk. 1995. Mercury in Siberia environment: estimation of contribution of natural
and anthropogenic origins. Novosibirsk, 1995. 30 p.
NTS "Tehnolog". 1990. About results of work on determination of mercury
contamination of industrial production of chlorine and caustic soda at Pavlodar
Chemical Plant. Pavlodar, Pavlodar hydrogeological expedition. NTS "Tehnolog",
1990. P. 41.
"Sinteko". 1992. Recommendations for environment protection from mercury
contamination. Kiev, KNII "Sinteko", 1992. P.22.
"Sintez". 1991.Initial data and recommendations to eliminate mercury contamination of
soils of the industrial production of chlorine and caustic soda on mercury method at
Pavlodar Chemical Plant. Kiev, KHIFMNPO " Sintez", 1991. 43 p.
Zhetekshi. 1993. Yearbook on regime and balance study of groundwater in the territory
of Pavlodar oblast for 1992-1993. Book 1. Zhetekshi. Pavlodar hydrogeological
expedition, 1993. P. 148.
Zhetekshi. 1991. Regime and balance of groundwater in the territory of Pavlodar oblast.
Book 1. Zhetekshi. Pavlodar hydrogeological expedition, 1991. P.233.
Zhetekshi. 1988. The control for groundwater protection to prevent the depletion and
contamination in the territory of Pavlodar oblast for 1986-1987. Book 1. Zhetekshi.
Pavlodar hydrogeological expedition, 1988. P.84.
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