〔461〕

Studies

on Microbial Synthesis and Decomposition of Organomercury Compounds Masahiko

Department

of Public Health,

Taira

School of Medicine, Kobe University,

Kobe

SYNOPSIS The studies conducted at Kumamoto University concluded that the Minamata disease is caused by methyl mercury. In the course of a nationwide survey made thereafter on environmental pollution by mercury, methyl mercury was detected in fish and human hairs which were supposedly free from artificial contamination by mercury. In particular, 60 to 70% of mercury found in tuna caught in oceans has been reported to be in the form of methyl mercury. An abnormally high mercury content in fish has also been reported in Sweden. Westoo carried out analyses of mercury in fish and other foodstuffs and claimed that 80 to 100% of mercury is present as methyl mercury. To elucidate the origin of such methyl mercury in nature and the effects of methyl mercury on mankind from the standpoint of public health, microbiological and ecological studies were undertaken on microbial conversion of inorganic mercury into organic mercury and of a process of the biological chain. A mercury-resistant bacterium belonging to Pseudomonas was separated from sewage water and biological synthesis and decomposition of organic mercury by this bacterium were investigated. Following this, microorganisms growing in a zone of mercury deposits, soils, rivers and seas and certain eumycetes were found to possess the ability to synthesize organic mercury. Hence, the possibility of conversion of inorganic mercury into organic mercury in nature was recognized. Furthermore, the extent of synthesis of organic mercury and the extent of accumulation of methyl mercury in fish by way of biological concentration were studied with the aid of ecological means. The microbially synthesized methyl mercury is concentrated by the food chain and an unusually high mercury content is sometimes found in fish. However, accumulation of methyl mercury in fish seldom proceeds to such a high level as to cause mercury poisoning, unless continual flow of methyl mercury in abnormally large quantities is present, as has been known in the case of the Minamata disease. Therefore, detection of an abnormally large amount of mercury in fish or shellfish suggests artificial contamination by mercury in one way or another, and a countermeasure must be worked out.

Chapter

1.

Synthesis

and

Compounds

1)

Decomposition by

a

of

Organomercury

Mercury-resistant

Bacterium

INTRODUCTION

It is known throughout the world that two incidences of the Minamata disease in Japan, one in and around the Minamata Bay and the other along the Agano River in Niigata Prefecture,1-3)produced a large number of mercury poisoned cases. Every one of these cases has been traced to contamination of the living environment by waste waters discharged from factories using mercury. In the process of synthesis of acetaldehyde that is carried out in

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the presence of mercury sulfate as a catalyst, methyl mercury is inevitably produced as a by-product.4-6) Waste water containing this methyl mercury was discharged from the factories into the Minamata Bay or the Agano River, thereby polluting the waters; the methyl mercury was in the meantime transferred to fish and shellfish and concentrated there by way of the food chain. Symptoms of mercury poisoning appeared in those people who ate such fish or shellfish in large quantities.7)

Later, the method of making this organic chemical product was changed from the conventional method starting from acetylene to a new method based on ethylene, and the environmental pollution by methyl mercury was brought under reasonably effective control.8) However, due to accumulation of mercury in the environment by continual artificial release to date,9) and furthermore due to the fact that Japan is situated in the circumpacific belt of mercury deposits, there are numerous regions in Japan where the mercury content in the soil far exceeds the Clarke number of 0.2ppm for mercury.10) In addition, the environmental pollution by alkyl mercury compounds has developed into a major social problem and a strenuous survey conducted in this country indicates that mercury in fish caught in those regions which are supposedly free from contamination by the artificially made alkyl mercury compounds is present in a large measure as methyl mercury.10-14) Similarly, in Sweden, Westoo maintains that 80 to 100% of mercury in fish is methyl mercury.14) Thus, mercury in fish is mostly in the form of methyl mercury, although the mercury compounds discharged into rivers are virtually all inorganic, or only traces of methyl mercury if they are organic. Moreover, Tanaka suggested a possibility of microbial conversion of inorganic mercury compounds into organomercury compounds followed by formation of CH3Hg-compounds in fish or shellfish in his probe into the mechanism of the Minamata disease.1) In the light of these findings, the author of this study assumed the existence of a mechanism for methylation of mercury in nature, and, in particular, a participation of microorganisms in such a mechanism. In this chapter, he first describes separation of a mercuric chloride-resistant bacterium, and then synthesis of organomercury compounds from mercuric chloride (conversion of inorganic mercury into organic mercury), and decomposition of a variety of alkyl mercury compounds by the mercury-resistant bacterium thus separated. 2) (1)

EXPERIMENTAL MATERIALS AND METHODS

Separation of Mercuric Chloride-resistant Bacterium. The basal culture medium was prepared by dissolving 5g of meat extract, 10g of poly-

peptone and 5g of sodium chloride in 1 liter of water and adjusting the pH at 7.0. After the addition of 100ppm of mercuric chloride, the liquid culture medium was added to the sample

collected

from

sewage

water

contaminated

with

mercury,

and

cultivated

at

37℃

for

4 days. The bacterium was selected from a grown colony and cultivated in a plate agar culture medium which had been prepared by adding agar to the same culture medium as stated above. Selection and cultivation were repeated several times with microscopic observation until pure separation of the bacterium in question was completed. (2)

Synthesis of Organomercury Compounds by the Mercury-resistant Bacterium. 1. Liquid culture media were prepared by adding 100, 200, 300 or 400ppm of mercuric chloride to the above-mentioned basal culture medium. Each culture medium (8ml) was introduced into a test tube which was 12mm in diameter, inoculated with the mercury-resistant

bacterium

in

the

usual

manner

with

the

aid

of

a

platinum

loop

and

cultivated

at

37℃.

The culture liquid was sampled at intervals for determination of organomercury compounds. 2. To the synthetic culture medium which had been prepared by dissolving 1.9g of Na2HPO4, 0.19g of NaH2PO4, 0.2g of MgSO4, 3g of (NH4)2SO4, 10g of glucose in 1l of

VOL.

water and adjusting further or

a

by

either

mixture

of

4, OCTOBER

the pH at 6.0, there

10μg/ml amino

30, NO.

of acids

was added 10ppm of mercuric chloride followed

L-methionine, (1μg/ml

1975〔463〕

10μg/ml each

of

of

aspartic

L-ethionine, acid,

0.1μg/ml

glutamic

of

acid,

vitamin

alanine,

B12

arginine,

cystine, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine and valine). Each culture medium thus prepared was transferred to a test tube which was 12mm in diameter, inoculated with a loopful of the mercury-resistant bacterium

in

the

usual

manner,

cultivated

at

37℃

and

sampled

at

intervals

for

determination

of organomercury compounds. (3) Decomposition of Organomercury Compounds by the Mercury-resistant Bacterium. 1. Liquid culture media were prepared by adding phenylmercuric acetate, methylmercuric chloride or ethylmercuric phosphate to the above-mentioned basal culture medium, to a specified concentration. Each liquid culture medium was transferred to a test tube which was 12mm in diameter, inoculated with a loopful of the mercury-resistant bacterium in the usual manner, cultivated

at

37℃

and

sampled

at

intervals

for

determination

of

organic

mercury

and

total

mercury. 2. Methylmercuric chloride, ethylmercuric chloride, butylmercuric chloride, propylmercuric chloride or amylmercuric chloride was added, to the final concentration of 1ppm, to a synthetic culture medium which had been prepared by dissolving 1.9g of Na2HPO4, 0.19g of NaH2PO4 0.2g of MgSO4, 3g of (NH4)2SO4, 10g of glucose and 0.5g of peptone in 1l of water and adjusting the pH at 6.0. The resulting medium was inoculated with a loopful of the

mercury-resistant

vals for determination

bacterium

in

of organic

the

usual

mercury

manner,

and total

cultivated

at

37℃

and

sampled

at

inter-

mercury.

(4)

The organomercury compounds were determined by gas chromatography15) and part by gas chromatography-mass spectroscopy. The total mercury was determined atomic absorption spectrophotometry.16)

(5)

The

degree of

was

determined

bacterial

by

the

growth was

Bertrand

3) Table

1.

shown

by

the optical

density

at

620mμ.

in by

Glucose

method.

EXPERIMENTAL RESULTS

Morpholohical and Physiological Characters of the Mercury-resistant Badterium

Table

2.

Bacteriostatic Mercuric

* the cell suspension to the

basal

concentrations incubated

minimam

Activity

of each

liquid

bacterium

medium

7

days

concentration

PMA, which arrested rium was checked. ** quoted from reference

at

added various

EMP, or PMA,

37℃.

of

was

containing

of MC, MMC, for

of

Compounds

After

MC, MMC,

the growth

and

then,

the

EMP,

of each

or

bacte-

19

MC: HgCl2, MMC: CH3HgCl, EMP: (C2H5Hg)2HPO4, PMA: C6H5Hg(CH3COO)

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(1)

Bacteriological Characters of the Marcury-resistant Bacterium and Bacteriostatic Concentration. The mercury-resistant bacterium separated from sewage water contaminated with mercury was identified in accordance with "Bergey's Manual of Determinative Bacteriology", 7th ed. (1957).17) The results are shown in Table 1. On the basis of morphological and physiological characters, the bacterium in question was identified as a species of Pseudomonas (Taira,1968).18) The resistance of this bacterium to phenylmercuric acetate, methylmercuric chloride and mercuric chloride was examined. The bacterium was added to the basal culture medium containing phenylmercuric acetate, methylmercuric chloride or mercuric chloride at varying

concentrations

and

cultivated

without

shaking

at

maximum concentration which allows bacterial growth. indicate that this bacterium is several thousand times bacteria.19)

37℃

for

7

days

to

determine

the

The results, shown in Table 2., as resistant to mercury as other

-●-100ppm -×-200ppm -○-300ppm -△-400ppm

-●-200ppm -○-300ppm

-×-400ppm

-●-200ppm -○-300ppm -×-400ppm

(Note) Medium: basal medium (bouillon containing 10mg of methionine per liter) containing 100, (Note) Medium: bouillon (8ml) containing 200, 300 or 400ppm Cuitivation

Amount

temperature:

of organic

1.

Synthesis pounds by bacterium

of

Amount shown

organomercury the

Cultivation

37℃

mercury:

mercury-resistant

temperature:

of organic

per 8ml

of medium

Fig.

200, 300 or 400ppm

of mercuric chloride.

mercury:

shown

per 8ml

of

medium

Fig. com-

of mercuric chloride.

37℃

2.

Synthesis

of

organomercury

com-

pounds by the mercury-resistant bacterium in the presence of methionine

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Synthesis of Organomercury Compounds by the Mercury-resistant Bacterium. 1. Effect of the HgCl2 Concentration of Microbial Synthesis of Organomercury pounds. The

bacterium

which

had

been

pre-cultivated

at

prepared by adding 100ppm of mercuric chloride by using a platinum loop in the usual manner chloride and cultivated. compounds with time

similar

also

the

above

were

made

with

the

for

48

hours

in

a

culture

medium

to the basal culture medium was inoculated to the basal culture medium containing a

given amount of mercuric synthesized organomercury to

37℃

Com-

The changes in the amount of microorgan are shown in Fig. 1. The experiments

addition

of

10μg/l

of

L-methionine

and

the

results are shown in Fig. 2. A comparison of Fig. 1 and Fig. 2 shows no significant differences in the amount of organomercury compounds synthesized when methionine was absent or present, but in both cases the amount of synthesized organic mercury tended to increase as the amount of mercury added to the culture medium increased. The maximum amounts of

synthesized

mercury

organomercury

chloride,

compounds

1.63μg/10ml

with

were

addition

1.75μg/10ml of

300ppm

With of

addition

mercuric

of

chloride

400ppm and

of

0.75μg/

10ml with addition of 200ppm of mercuric chloride. In each case, ethyl mercury was synthesized in an amount about 1/3 of that of methyl mercury. On the other hand, in the presence of

of

methionine,

synthesized

1.54μg/10ml

the

maximum

organomercury with

the

addition

amount

compounds of

400ppm

Table

3,

Effect

was of

mercuric chloride. 2. Effect of the Cultivation Temperature on Microbial Synthesis of Organomercury Compounds. Thee effect of the cultivation temperature on the microbial synthesis of organomercury compounds was studied using the basal culture medium containing 10ppm of mercuric chloride. The results are shown in Table 3 and they indicate that the synthesis proceeds best at

of Cultivation

on Synthesis Compounds

mediun:

peptone 10g, glucose 1g, meat extract 10g, NaCl 5g, HgCl2 (as Hg) 10mg, water 1,000ml

4.

Effects of the Initial the medium on the Organomercury

● ○

Hg++ Hg++

10ppm

Hg++

100ppm

ture:

Medium: 3.

pH values Synthesis

of of

Compounds

0ppm

* Incubation

Fig.

Temperature Organomercury

* cultivation time: 10 days

Table

×

of

Change during Bacterium

of the Culture

Medium pH Values of Mercury-resistant

times:

7 days,

Incubation

tempera-

37℃

Peptone 10g, meat extract 10g, glucose 1g, NaCl 5g, HgCl2 (as Hg) 10mg, water 1,000ml MM: methyl mercury, EM: ethyl mercury

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and

JOURNAL evidently

much

less

below

10℃

or

OF above

HYGIENE 40℃.

3. Effect of the pH on Microbial Synthesis of Organomercury Compounds. The effect of the initial pH of the culture medium on the microbial synthesis of organomercury compounds was studied and the results are shown in Table 4. It is apparent that the synthesis proceeds more readily in the acidic range. The changes in the pH of the culture medium with time during bacterial growth are shown in Fig. 3. The pH falls to distinctly different levels depending upon whether mercuric chloride has been added or not, and this behavior suggests a possible change in metabolism as a result of the addition of mercuric chloride. 4. Effect of Methionine, Ethionine, Vitamin B12 and Amino Acids on Microbial Synthesis of Organomercury Compounds. To the above-mentioned synthetic culture medium was added 10ppm of mercuric chloride, which was then divided into five groups, followed further by L-methionine as a donor of the methyl group for Group II, L-ethionine as a donor of the ethyl group for Group III, vitamin B12 as a co-factor for transfer of the methyl group for Group IV or a mixture of amino acids (however, not containing methionine or ethionine) for providing better nutrient conditions for Group V. The bacterium pre-cultivated in the above-mentioned manner were inoculated by a platinum loop in the usual manner, cultivated and sampled at regular intervals for determination of the synthesized organomercury compounds. The results are shown in Table 5. Groups I, II, III, IV and V showed an occurrence of microbial synthesis of methyl mercury and etheyl mercury, but no marked differences in the amount of organomercury compounds synthesized were produced among the groups. Table

5.

Effects of various substances compounds by Mercury-resistant

on synthesis Bacterium

of organomercury (μg/ml

MM:

(3)

methyl

mercury,

EM:

ethyl

as Hg)

mercury

Decomposition of Organomercury Compounds by the Mercury-resistant Bacterium. The occurrence of decomposition of phenylmercuric acetate by the mercury-resistant bacterium was examined. With the addition of 100ppm of phenylmercuric acetate, the bacterial growth was inhibited and hence no decomposition of phenylmercuric acetate was

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observed. On the other hand, the decomposition of phenylmercuric acetate proceeded very rapidly with the addition of 10ppm or 1ppm of phenylmercuric acetate. As for a decrease in total mercury, however, the samples here did not show any differences from the controls. The progress of decomposition with the addition of 10ppm or 1ppm of phenylmercuric acetate are shown in terms of percentages in Fig. 4-1. The broken line shows how mercury decreases in the absence of the bacterium. Similarly, the decompositions of methylmercuric chloride and ethylmercuric phosphate are shown in Figs. 4-2 and 4-3, respectively. Next, various alkyl mercury compounds were subjected, at a concentration level of 1ppm, to the decomposing action of the mercury-resistant bacterium in the above-mentioned synthetic culture medium. The results are shown in Fig. 5. Both consumption of glucose and bacterial growth were observed at the same time. The decomposition of organomercury compounds was found most vigorous in the logarithmic growth phase of the bacterium in question. Fig. 4-1

Decomposition

of PMA by Mercury-resistant

Fig. 4-2

Decomposition

of MMC by Mercury-resistant

Fig. 4.3

Decomposition

Fig.

4.

Decomposition

of EMP

by Mercury -resistant

of Organomercury

by Mercury-resistant

Bacterium

Bacterium

Bacterium

Bacterium

Compounds

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○

MM

(

●

EM

(ethyl

○

ProM

●

BM

methyl

● mercury)

× mercury)

(prophyl

mercury)

● ○

Fig.

5.

Decomposition Mercury-resistant

of Various Organomercury Bacterium

(buthyl mercury)

Compounds

by

(4)

Synthesis and Decomposition of Organomercury Compounds by the Cell Homogenate of the Mercury-resistant Bacterium. The bacterium was pre-cultivated for 3 days in the above-mentioned basal culture medium containing 100ppm of mercuric chloride, then cultivated for 24 hours in the basal culture medium containing no mercury, collected, washed with a 1/15M phosphate buffer solution (pH 7) containing 0.01M of glucose, suspended (1g/10ml) in the 1/15M phosphate buffer solution and homogenized by a cell homogenizer. Synthesis and decomposition of organomercury compounds by the cell homogenate thus prepared are shown in Table 6 and Fig. 6, respectively. 4)

DISCUSSION

Jernelov et al. found that methyl mercury was formed when mercuric chloride was added to the bottom sediments of a lake and left there for 5 to 10 days, and that mercury was similarly methylated to methyl mercury and more volatile dimethyl mercury by the action

VOL. Table

*

reaction Methionin

**

reaction

6.

mixture:

Synthesis

1/15M

10μg/10ml, temp.:

30, NO.

4, OCTOBER

1975〔469〕

of MMC by Mercury-resistant

phosphate Choline

buffer

10ml,

10μg/10ml,

1/100M

Bacteria

Bacteria

glucose, homogenate

Homogenate

1/100M

ATP,

VB

12 10μg/10ml,

1g/10ml

30℃

*

reaction

mixture

Table

…

1/15M phosphate buffer.

7.

Amounts of the synthesized MM by chemical reaction of Hg++ and methyl

1/100M glucose

cobalamine

1/100M ATP Bacteria homogenate (0.1g/ml) CH3HgCl

(1μg/ml)

reaction time (hours) **

remaining original

Fig.

6.

CH

3Hg-/

×100%

CH3Hg-

Decomposition of MMC by Mercuryresistant Bacteria Homogenate

Incubations were performed in brown ten ml of water solution were extracted. determined by ECD and before and after purification Values (-) mean

MM:

in this table below

methyl

were

flask and MM was

mass fragmentgraphy of cysteine. obtained

by ECD

and

0.01μM.

mercury

of a homogenate of dead fish. They also found, however, that the ability to methylate mercury was lost when the bottom sediments were sterilized in advance. On the basis of these findings, Jernelov et al. suggested a participation of microorganisms in this reaction.20,21) Moreover, Wood et al. reported that methane was generated from the methyl group of methylcobalamin in an atmosphere of hydrogen when ATP and methylcobalamin were added to the

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cell extracts of a methanogenic bacterium and, if Hg++ was present then, the CH3 group of methylcobalamin moved from Co to Hg to form methyl mercury.22) The author has also conducted experiments on purely chemical methylation of Hg++ by methylcobalamin (shown in Table 7). The synthesis of methyl mercury is affected simply by bringing methylcobalamin into contact with HgCl2 in an aqueous solution, but this methylation reaction is inhibited by the addition of cysteine to the solution.23) Bertilsson et al. reported on chemical methylation by methylcobalamin, but cast some doubt on an occurrence of methylation by methylcobalamin in the living body in the light of marked inhibition of this reaction by amino acids and proteins containing the -SH group.24) It is therefore difficult to assess at the present time how far the non-enzymatic methylation reaction such as this might occur in the living body or in the ecological system. In addition to mercury, methylation also occurs in As, Se and Te.25,26) Methylation of As is effected by such eumycetes as Aspergillus, Mucor, Penicilluium, Fusarium and Paecilamyces and this methylation reaction yields monomethyl-, dimethyl- and trimethylarsine by way of stepwise transfer of the methyl group from methionine or its related substances to arsenous acid.25) Methylation of arsenate which occurs in dimethylarsine involving methylcobalamin is also known. It this case, however, the reaction proceeds under anaerobic conditions requiring the presence of ATP and molecular hydrogen and is a reductive methylation in which methylcobalamin serves as a donor of the methyl group.26) Similarly to the microbial synthesis of trimethylarsine from arsenite or arsenate, dimethylselene is synthesized from sodium selenate or sodium selenite.25) These findings suggest the following systems for methylation of mercury. i) A system involving S-adenosylmethionine; introduction of the methyl group to homocysteine gives rise to formation of methionine, but if mercury is present in this system, the formation of methionine is inhibited and methyl mercury is formed instead. ii) A system involving methylcobalamin; transfer of the methyl group from Co in methylcobalamin to mercury yields methyl mercury. iii) The methyl group in methionine or related substances participates via transfer in the formation of methyl mercury. Now, the bacterium the author separated is an aerobe, and furthermore it does not promote methylation even with the addition of vitamin B12. Moreover, cysteine is known to inhibit direct methylation of Hg++ by methylcobalamin.23) Hence it is difficult to accept the occurrence of methylation by the second system. The addition of methionine causes no marked increasee in the amount of methyl mercury synthesized, but reduces by half the time reguired to reach a peak in microbial syntheses. Furthermore, a strain of Neurospora crassa which has acquired resistance to mercury by X-ray irradiation produces methyl mercury when cultivated in a medium containing homocysteine or cysteine and mercuric chloride. Vitamin B12does not participate in the metabolic system of this Neurospora crassa.27) Therefore, the presence of a methyl donor other than methylcobalamin is conceivable for methylation of mercury by this bacterial strain. Hence, the author sees sufficient ground to assume that methionine is related in one way or another to the microbial synthesis of methyl mercury occuring by the first or third system. At any rate, the author believes that he has here sufficiently proved experimentally an occurrence of microbial synthesis of organomercury compounds from mercuric chloride. However, the same microorganism simultaneously converts organomercury compounds into inorganic mercury compounds. A comparison of the rate

of

rate

of

microbial decomposition

that

the

decomposition

synthesis of

of

methyl

methyl

proceeds

mercury

mercury about

7.4×104

shown shown times

in in

Fig.

Fig. as

fast

4-2 as

1

(5.7×10-7μg/ml/hr)

with

(42×10-2μg/ml/hr)

indicates

the

synthesis.

The

the

mechanism

of this decomposition has been investigated in detail by Tonomura et al. When bacteria of Pseudomonas are added to a solution of phenyl mercury, a large quantity of phenyl mercury becomes bound to the cell surface first, the bound phenyl mercury is _reduced to metallic

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mercury and the metallic mercury vaporizes out of the system. The addition of a compound containing the -SH group is needed for this decomposition; this compound first binds itself with the organomercury compound and the decomposition reaction follows thereafter.28,29) The decomposition occurs in conjunction with glucose dehydrogenase which supplies reduced NAD (P). It appears likely that organomercury compounds are decomposed in a pathway similar to the respiratory system involving cytochrome C-1, the one with higher molecular weight of the two kinds of cytochrome C.30) When radioactive mercuric chloride is mixed with a non-radioactive organomercury compound, the exchange reaction of mercury takes place quite rapidly to yield a radioactive organomercury compound. This demonstrates that the C-Hg linkage in organomercury compounds is not very strong. Moreover, the C-Hg linkage tends to weaken as the number of C bound to mercury increases.19,31) This can also be inferred more or less from the author's decomposition experiments. However, environmental factors have not been considered in this comparison of microbial conversion of organic mercury into inorganic mercury and microbial conversion of inorganic mercury into organic mercury. An occurrence of conversion of inorganic mercury into organic mercury at a relatively high velocity under certain environmental conditions may be presumed from the fact that methyl mercury is found in fish caught in regions which are not contaminated with artificially made organomercury compounds. A proof of in vitro synthesis of organomercury compounds from inorganic mercury by pure microorganisms merely suggests a possibility of occurrence of such synthesis in nature. A wide variety of microorganisms grow in nature, forming aggregates and affecting each other but do not occur alone. Moreover, the natural environment changes from moment to moment under the influence of chemical and physical agencies. What reaction will proceed how fast in what environment is of great significance when one considers the problems of mercury from the standpoint of public health. This subject will be taken up in Chapter 2. 5)

CONCLUSIONS

A mercury-resistant bacterium belonging to Pseudomonas was obtained from sewage water contaminated with mercury and the action of this bacterium on mercury was investigated. The results are as follows: (i) The mercury-resistant bacterium was capable of synthesizing methyl mercury and ethyl mercury from mercuric chloride. (ii) The mercury-resistant bacterium decomposed phenylmercuric acetate and other alkyl mercury compounds (methyl mercury, ethyl mercury, propyl mercury, butyl mercury and amyl mercury). The decomposition proceeded most vigorously in the logarithmic growth phase. (iii) The cell-free extract of the mercury-resistant bacterium synthesized methyl mercury from mercuric chloride. This extract also decomposed methyl mercury. However, fractional extraction of the enzyme having both synthesizing and decomposing abilities was not successful. (iv) With the mercury-resistant bacterium, conversion of organic mercury to inorganic mercury

or

mercury

to organic

decomposition

proceeded

mercury

Chapter

2.

about

Confirmation Compounds 1)

The

Minamata

disease

7.4×104

times

as

fast

as

conversion

of

inorganic

or synthesis.

is caused

of Synthesis Occurring

of Organomercury in Nature

INTRODUCTION

by methyl

mercury

and the

hazardous

nature

of lower

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alkyl mercury compounds is universally known.6,7) However inorganic mercury has not yet been duly recognized as a possible health hazard. As the author made clear in Chapter 1, there are microorganisms which are capable of methylating inorganic mercury and as Jernelov of Sweden pointed out, a mechanism is at work to methylate mercury in certain environments.20,21) Attention should therefore be paid to the discharge of inorganic mercury from factories into the natural environment and to leaching of mercury from mercury mines and mercury deposits. Now, the in vitro methylation of mercury by purely cultivated bacteria which the author described in Chapter 1 merely suggested a possibility of the same process happening in nature. Consequently, in this chapter, the author studied conversion of inorganic mercury into organic mercury by microorganisms growing in different environments (various eumycetes, in river waters, sea waters, soils of mercury mines and ordinary soils) and examined the possibility of microbial synthesis of methyl mercury in nature. 2) (1)

EXPERIMENTAL MATERIALS AND METHODS

Synthesis of Methyl Mercury by Various Eumycetes and Activated The following strains were used:

Sludge.

(a) Neurospora crassa (b) Penicillium notatum (c) Aspergillus niger (d) Mucor mucedo (e) Rhizopus stoloniter (f) Plants treated with sewage sludge The strain was inoculated to the Sabouraud liquid glucose medium containing mercuric

chloride,

cultivated

at

27℃,

and

the

liquid

was

sampled

at

regular

5ppm of

intervals

for

determination of organic mercury. On the last day of cultivation, the liquid was separated from the bacteria, and organic mercury and total mercury were determined. As for the experiments with the activated sludge, the activated sludge and HgCl2 were added to a culture medium which had been prepared by dissolving 100mg of K2HPO4, 20mg of CaCl2, 20mg of MgSO4, 10mg of FeCl2 and 100mg of (NH4)2SO4 in 1l of water, so, so that the contents of the activated sludge and HgCl2 became 2,000ppm and 200ppm, respectively. Further, 100ppm of glucose, 400ppm of methionine and 600ppm of crotonic acid were added daily and the amounts of synthesized organic mercury were determined at various time intervals. (2)

Synthesis of Methyl Mercury Waters.

Organic mercury Gram negative bacilli spectively inoculated taining

mercuric

by Microorganisms

Growing

in River Waters and Sea

and total mercury in river mud and algae were determined and the separated from these and bacteria separated from seashore were reinto the same basal culture medium as mentioned in Chapter 1 con-

chloride,

cultivated

at

37℃,

and

ahalyzed

for

organic

mercury

thereby

synthesized. (3)

Synthesis of Methyl Mercury by Microorganisms in Soils. Mud sampled in a certain mercury refinery was analyzed for total mercury and a part of it (0.1g) was inoculated into the same basal culture medium as mentioned in Chapter 1, containing mercuric chloride, cultivated under aerobic conditions as well as under anaerobic conditions (in an apparatus shown in Fig. 7) and analyzed for organic mercury thereby microbially synthesized. Five grams of soil taken from the grounds of the Medical Department, Kobe University (total mercury 2.4ppm and methyl mercury 0.0016ppm on dry weight basis) was suspended

VOL.

30, NO.

4, OCTOBER

1975〔473〕

in 10ml of a phosphate buffer solution, cultivated after the addition of various given amounts of mercuric chloride, 100mg of peptone and 100mg of glucose and analyzed at regular intervals for organic mercury microbially synthesized. Furthermore, 30g of soil was placed in an ap-

paratus shown in Fig. 8, mercuric chloride was added to a given concentration, peptone and glucose, 1g each, were added every week and cultivation was carried out at room temperature. The supernatant liquid was sampled at regular intervals and analyzed for organic mercury microbially synthesized. (4) Mercury was analyzed in accordance with the methods described in Chapter 1.

Fig.

7.

Apparatus

for anaerobic

cultivation

3) (1)

8.

Synthesis pounds

Com-

Table

9.

Crassa

cultivation

MMC:

medium, temp:

CH3HgCl,

S:

slant medium,

27℃, -:

EMC: