Organomercurial-volatilizing bacteria in the mercury-polluted sediment of Minamata Bay, Japan.

A total of 4,604 bacterial strains isolated from the sediments of Minamata Bay and nearby low-level-mercury stations (control stations) were screened for the ability to volatilize mercury from inorganic and organic mercurial compounds. The strains that volatilize mercury from several kinds of organomercurials were found only in the sediments of Minamata Bay.     

Mercury pollution in Tokuyama Bay

The sediments and aquatic life of Tokuyama Bay, Japan, have been polluted by mercury effluent from chloro-alkali plants. In total, about 380 tons mercury were released from these plants and 6.64 tons of mercury were discharged into the bay in waste waters between 1952 and 1975, when mercury cells were employed. A number of surveys to study mercury pollution and the effectiveness of control measures in this area were conducted in the early 1970's by our laboroatory and other agencies. Analysis of human hair from Tokuyama Bay residents contained less mercury than those in Minamata and Agano districts, Japan, where serious mercury poisoning had occurred, but were contaminated with more mercury than those in other unpolluted areas. No occurrence of Minamata disease has been reported in the Tokuyama district.Reclamation of mercury contaminated sediments began in 1975; dredging of the bay continued until 1977. Since then, the levels of mercury contamination in sediments and aquatic life have gradually decreased. Today there are no problems with respect to mercury pollution.In this paper, we describe and discuss mercury pollution in Tokuyama Bay with regard to the following aspects of research and pollution control: the history of mercury pollution; mercury discharge and its accumulation in sediments; behaviour of mercury in sediments; mercury contamination of fish; mercury and the health of local residents; and remedial actions.     

Removal of mercury from mercury-contaminated sediments using a combined method of chemical leaching and volatilization of mercury by bacteria

A method for the removal of mercury sulfide frommercury-contaminated sediments was developed, whichconsists of chemical leaching and volatilization ofmercury by bacteria. More than 85% of the mercury insediment containing 0.11–37.4 mg/kg of mercury wasefficiently extracted with 3 M HCl and 74 mMFeCl₃. Subsequent volatilization by bacteriaresulted in the removal of 62.9–75.1% of mercury frommercury-contaminated Minamata Bay sediments. Methylmercury was also eliminated from soil at a highefficiency. Thus, this combined method of chemicaland microbial treatments could be used for efficientremoval of both organic and inorganic mercurials fromnatural sediments.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment.

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Role of plasmids in mercury transformation by bacteria isolated from the aquatic environment

Eight mercury-resistant bacterial strains isolated from the Chesapeake Bay and one strain isolated from the Cayman Trench were examined for ability to volatilize mercury. Mercury volatilization was found to be variable in the strains tested. In addition, plasmids were detected in all strains. After curing, two of the bacterial strains lost mercury resistance, indicating that volatilization is plasmid mediated in these strains. Only two cultures demonstrated ability to methylate mercuric chloride under either aerobic or anaerobic conditions. Methylation of mercury, compared with volatilization, appears to be mediated by a separate genetic system in these bacteria. It is concluded that mercury volatilization in the estuarine environment can be mediated by genes carried on plasmids.     

Mercury-resistant bacterial strains Pseudomonas and Psychrobacter spp. isolated from sediments of Orbetello Lagoon (Italy) and their possible use in bioremediation processes

This study was aimed to isolate Hg-resistant bacteria from contaminated sediments of the Orbetello Lagoon in Italy and to assess their possible use as biofilms in bioremediation processes. Enrichment cultures prepared from contaminated sediments in the presence of 0.05 mM of mercury and under aerobic conditions allowed the isolation of five heterotrophic bacterial strains. 16S rDNA gene sequencing assigned the isolated strains to the genera Pseudomonas and Psychrobacter. For the first time mercury-resistant bacterial strains belonging to the genus Psychrobacter were evidenced. Minimum inhibitory concentrations in the presence of HgCl₂ and of CH₃HgCl showed high levels of resistance. EC50 values for the isolated bacterial strains in the presence of HgCl₂ and of CH₃HgCl confirmed the adaptation to the metal. Hg-resistant strains ORHg1, ORHg4 and ORHg5 showed the capacity to volatilize inorganic and organic mercury to elemental mercury, and formed biofilms on pumice particles, and were shown to play a role in the removal of mercury from sediment leachates. This study reports isolation and characterization of new Hg-resistant bacterial strains and provides novel insight into their possible use in bioremediation processes of mercury polluted sediments.     

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.     

Mercury and Organomercurial Resistances Determined by Plasmids in Pseudomonas

Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.     

Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay

Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan. Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg(2+) and organomercurials) and narrow-spectrum (resistant to inorganic Hg(2+) and sensitive to organomercurials) groups. Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1. On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B. megaterium MB1 mer determinant. Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus. The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones.     

Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan.

A unique transposon was found in the chromosome of Bacillus megaterium MB1, a Gram-positive bacterium isolated from mercury-polluted sediments of Minamata Bay, Japan. The transposon region of a 14.5kb DNA fragment was amplified by PCR using a single PCR primer designed from the nucleotide sequence of an inverted repeat of class II transposons. The molecular analysis revealed that the PCR-amplified DNA fragment encodes a transposition module similar to that of Tn21. The transposon also encodes a broad-spectrum mercury resistance region having a restriction endonuclease map identical to that of Bacillus cereus RC607, a strain isolated from Boston Harbor, USA. The result of a phylogenetic analysis of the amino acid sequence of putative resolvase of the transposon showed that the transposon is phylogenetically closer to the transposons of Gram-positive bacteria than those of Gram-negative bacteria. Besides the transposition module and mer operon, the transposon encodes a mobile genetic element of bacterial group II introns between the resolvase gene and mer operon. The intron, however, does not intervene in any exon gene. The discovery of this newly found combination of the complex mobile elements may offer a clue to understanding the horizontal dissemination of broad-spectrum mercury resistance among microbes.     

Acanthamoeba stevensoni N. Sp. (Protozoa: Amoebida) from Sewage-Contaminated Shellfish Beds in Raritan Bay, New York

Marine sediments from 12 shallow water stations in Raritan Bay, New York were tested for the presence of Acanthamoeba. Eight stations were positive for one or more species of Acanthamoeba, A. castellanii, A. comandoni, A. hatchetti, A. lenticulata, A. polyphaga, A. rhysodes, and Acanthamoeba spp. Isolates that grew at 38–40° C were found at four stations (A. comandoni, A. lenticulata, and two unidentified strains). The two unknown strains were characterized on the basis of morphological features, isoenzyme profiles, and mouse pathogenicity tests. One of the two strains was determined to be a new species and is designated herein as Acanthamoeba stevensoni n. sp., ATCC 50388. Mature cysts were most similar to those of morphological Group II of Pussard & Pons (1977). Acanthamoeba stevensoni n. sp. was isolated from inshore coastal sediments where seawater ranged from 20–30%‰ (ppt.). The sediments supported commercially valuable populations of hard clams, Mercenaria mercenaria, that required depuration prior to sale because of contamination by sewage-associated bacteria.     

Isolation and Diversity of Actinomycetes in the Chesapeake Bay

Chesapeake Bay was investigated as a source of actinomycetes to screen for production of novel bioactive compounds. The presence of relatively large populations of actinoplanetes (chemotype II/D actinomycetes) in Chesapeake Bay sediment samples indicates that it is an eminently suitable ecosystem from which to isolate actinomycetes for screening programs. Actinomycetes were isolated from sediment samples collected in Chesapeake Bay with an isolation medium containing nalidixic acid, which proved to be more effective than heat pretreatment of samples. Actinomycete counts ranged from a high of 1.4 × 10⁵ to a low of 1.8 × 10² CFU/ml of sediment. Actinomycetes constituted 0.15 to 8.63% of the culturable microbial community. The majority of isolates from the eight stations studied were actinoplanetes (i.e., chemotype II/D), and 249 of these isolates were obtained in a total of 298 actinomycete isolates. Antimicrobial activity profiles indicated that diverse populations of actinoplanetes were present at each station. DNA hybridization studies showed considerable diversity among isolates between stations, but indicated that actinoplanete strains making up populations at nearby stations were more similar to each other than to populations sampled at distant stations. The diversity of actinoplanetes and the ease with which these organisms were isolated from Chesapeake Bay sediments make this a useful source of these actinomycetes.     

Detoxification of mercury and organomercurials by nitrogen-fixing soil bacteria

Minimal inhibitory concentration values of HgCl₂ and 5 organomercurials were determined against 24 mercury-resistant N₂-fixing soil bacteria previously isolated from soil and identified in our laboratory. These bacterial strains also displayed multiple antibiotic resistant properties. Typical growth pattern of a highly mercury-resistantBeijerinckia sp (KDr₂) was studied in liquid broth supplemented with toxic levels of mercury compounds. Four bacterial strains were selected for determining their ability to volatilize mercury and their Hg-volatilizing capacity was different. Cell-free extracts prepared from overnight mercury-induced cells catalyzed Hg²⁺-induced NADPH oxidation. Specific activities of Hg²⁺-reductase which is capable of catalyzing conversion of Hg²⁺ →Hg(o) of 10 Hg-resistant bacterial strains are also reported.     

Abundance and germination capability of resting cysts of Alexandrium spp. (Dinophyceae) from faecal pellets of macrobenthic organisms

The Alexandrium spp. resting cysts were found abundantly in faecal pellets collected from the bottom sediments at two stations in Hiroshima Bay. It is considered that these faecal pellets were excreted by the macrobenthos, such as polychaeta and mollusca, based on their size and morphology. Polychaeta was the most dominant macrobenthos, and mollusca was the second most dominant group in Hiroshima Bay. The resting cysts of Alexandrium spp. in the bottom sediments at the two stations were counted in both the faecal pellets of macrobenthos and in the surrounding sediment. As a result, the number of cysts in the faecal pellets accounted for 28.9-35.2% of total cysts. In addition, cysts isolated from faecal pellets had almost the same germination ability as those in the sediment. Thus, Alexandrium cysts are tolerant to the predation and digestive processes of macrobenthic organisms.     

Diversity of Archaea in marine sediments from Skan Bay, Alaska, including cultivated methanogens, and description of Methanogenium boonei sp. nov

Methanogenesis in cold marine sediments is a globally important process leading to methane hydrate deposits, cold seeps, physical instability of sediment, and atmospheric methane emissions. We employed a multidisciplinary approach that combined culture-dependent and -independent analyses with geochemical measurements in the sediments of Skan Bay, Alaska (53 degrees N, 167 degrees W), to investigate methanogenesis there. Cultivation-independent analyses of the archaeal community revealed that uncultivated microbes of the kingdoms Euryarchaeota and Crenarchaeota are present at Skan Bay and that methanogens constituted a small proportion of the archaeal community. Methanogens were cultivated from depths of 0 to 60 cm in the sediments, and several strains related to the orders Methanomicrobiales and Methanosarcinales were isolated. Isolates were psychrotolerant marine-adapted strains and included an aceticlastic methanogen, strain AK-6, as well as three strains of CO(2)-reducing methanogens: AK-3, AK7, and AK-8. The phylogenetic positions and physiological characteristics of these strains are described. We propose a new species, Methanogenium boonei, with strain AK-7 as the type strain.     

Sublittoral Macrobenthic Infaunal Assemblages of Two Nearby Embayments from Central Chile

The benthic assemblages in two Central Chile nearby embayments were studied from quantitative samples collected from 15 sites at depths of 8–65 m. The macrobenthic infauna (<0.5 mm) of both bays was greatly dominated by polychaetes. Some 93 taxa were identified, of which 51 were polychaetes. The average macrofaunal abundance for all stations (15,021 ind. m⁻²) is very close to the values reported for the neighboring areas. Numerical classification and ordination (DCA) of sites resulted in three site-groups mostly reflecting differences in the bottom sediments: the muddy-bottom stations of Concepción Bay and the shelf-associated stations, the sandy-bottom stations of San Vicente Bay and an heavily polluted station at San Vicente port. Classification of species showed that the muddy-bottom stations and the sandy-bottom sites had characteristic species assemblages. The macrofaunal assemblages presented high dominance values, which were due to the high numerical abundances of a few species in the collections.     

Heavy metal contamination of sediments in the Upper Connecting Channels of the Great Lakes

In 1985, sampling at 250 stations throughout the St. Marys, St. Clair, and Detroit rivers and Lake St. Clair — the connecting channels of the upper Great Lakes — revealed widespread metal contamination of the sediments. Concentrations of cadmium, chromium, copper, lead, mercury, nickel, and zinc each exceeded U.S. Environmental Protection Agency sediment pollution guidelines at one or more stations throughout the study area. Sediments were polluted more frequently by copper, nickel, zinc, and lead than by cadmium, chromium, or mercury. Sediments with the highest concentrations of metals were found (in descending order) in the Detroit River, the St. Marys River, the St. Clair River, and Lake St. Clair. Although metal contamination of sediments was most common and sediment concentrations of metals were generally highest near industrial areas, substantial contamination of sediments by metals was present in sediment deposition areas up to 60 km from any known source of pollution.Contribution 735 of the National Fisheries Research Center-Great Lakes, U.S. Fish and Wildlife Service, 1451 Green Road, Ann Arbor, MI 48105.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Metal resistance and accumulation in bacteria

Recent research on the ecology, physiology and genetics of metal resistance and accumulation in bacteria has significantly increased the basic understanding of microbiology in these areas. Research has clearly demonstrated the versatility of bacteria to cope with toxic metal ions. For example, certain strains of bacteria can efficiently efflux toxic ions such as cadmium, that normally exert an inhibitory effect on bacteria. Some bacteria such as Escherichia coli and Staphylococcus sp. can volatilize mercury via enzymatic transformations. It is also noteworthy that many of these resistance mechanisms are encoded on plasmids or transposons. By expanding the knowledge on metal-resistance and accumulation mechanisms in bacteria, it may be possible to utilize certain strains to recover precious metals such as gold and silver, or alternatively remove toxic metal ions from environments or products where their presence is undesirable.     

Volatilization of mercury byThiobacillus ferrooxidans

Thiobacillus ferrooxidans became significantly more tolerant to mercury stress after culturing in media of increasing mercury(II) concentrations. When mercuric chloride was added to the growth medium, the resistant organisms were found to volatilize elemental mercury (Hg⁰).T. ferrooxidans may be an important factor in the natural mercury cycle, since the environments whereT. ferrooxidans is found typically contain elevated levels of heavy metals, including mercury.