Heavy metals detoxification by mercury-resistant bacteria

Heavy metal pollution in industrial areas is a serious environmental concern and metal-resistant microorganisms have been already isolated from various polluted environments. In this study, 13 marine mercury-resistant bacterial strains have been isolated from various locations along the Indian Coast, using seawater nutrient agar (SWNA) amended with various HgCl₂ concentrations. Then, bacterial colonies capable of growth with more than 25 ppm of mercury were termed by the authors Bacteria Highly Resistant to Mercury (BHRM) and identified through biochemical and 16S rRNA gene sequence analyses as: Alcaligenes faecalis (7 isolates: GO01, GO02, GP06, GP14, GP15, GP16 and GP17), Bacillus pumilus (3 isolates: GP08, CH13 and S3), Pseudomonas aeruginosa (CH07), Brevibacterium iodinium (GP13) and Bacillus sp. (CM10).

Subsequently, each of these isolates has been tested by the authors in order to evaluate their resistance and ability to detoxify Cd, Pb and Hg. Mechanisms of metal resistance and detoxification in microbes include precipitation of metals as phosphates, carbonates or sulfides (for Pb); volatilization (for Hg) via methylation or ethylation; energy-dependent metal efflux systems, intracellular sequestration with cysteine-rich proteins and physical exclusion or entrapment of electronegative components in membranes and extracellular polymeric substances (EPS).

In order to evaluate Hg volatilization, cells were transferred by the authors to plates enriched with 10 ppm Hg. The plates were then covered with a Kodak XAR film and incubated. All of the tested isolates were capable of volatilizing mercury, as indicated by fogging of the film covering the plates (reduction of AgCl₂ by gaseous Hg); while a negative control did not volatilize Hg. It is well-known that the ability of volatilizing mercury is conferred by the highly conserved mer operon. Thus, in order to confirm his presence in the isolates, the authors extracted genomic DNA from the cultures and then amplified and visualized the merA region through PCR and gel electrophoresis. Surprisingly, the analyses revealed the presence of merA gene fragments only in 9 of the BHRM isolates, whereas all of them were volatilizing Hg. Thus the authors hypothesized the presence of another unknown non-mer mediated mercury volatilization mechanism in two isolates (CH13 and CM10), but this interesting possibility remains to be confirmed.

To estimate Cadmium detoxification instead, two isolates (CH07, GP06) were tested with different concentrations of Cd (as CdCl2) and metal concentrations were determined by inductively coupled plasma-atomic emission spectrometry. Results showed that isolate CH07 removed Cd from the medium at a faster rate than GP06. Cd concentration was reduced from an initial concentration of 100 ppm to 17.4 ppm (75%) by CH07 and to 19.2 ppm (70%) by GP06 in about 72 h. Several mechanisms of Cd resistance have been already described, but according to the authors, the observed metal accumulation in bacterial cell pellets is perhaps consistent with some kind of microbial bioabsorption of the metal.

Finally, regarding lead detoxification, 3 BHRM isolates (CH07, GP13 and S3) were tested by the authors with different concentrations of Pb (as (CH3COO)2Pb) using an experimental procedure similar to that used for Cd. Results showed that CH07 was able to reduce Pb concentration from 100 ppm to 1.8 ppm (>98% removal) in 96 h, while isolates GP13 and S3 removed more than 87% and 88% of Pb respectively from the growth medium. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) analysis showed that Pb was entrapped in the exopolymeric substance (EPS) in the case of isolate CH07 (negatively charged EPS in fact, could bind lead and prevent its entry into the cell), while hydrogen sulfide production suggested that Pb was most likely precipitated as sulfide by isolates GP13 and S3. In addition, EDS analysis of the cells showed the concentrations of Pb and Cd to be as high as 21% and 19%, respectively, while bacteria grown in medium without heavy metals showed no signals for either Cd or Pb. Moreover, the morphology of isolate GP13 was unchanged after growth in medium with 50 ppm Pb, suggesting thus, that Pb is not toxic to the isolate under the conditions tested.

Various metal-resistance mechanisms has been already found and characterized at the molecular level in several other bacterial strains. Moreover, it has been shown that bacterial mobile genetic elements, such as plasmids or transposons, can carry multiple genes encoding for metal resistance. Thus, exposure to one agent may select for microorganisms resistant to several toxicants. So, it is likely that multi-metal resistant BHRM, possess the genetic components for dealing with many toxic metal ions (including Hg, Cd, Zn, Sn, Cu, and Pb) and removing them from the surrounding medium using several high-efficiency processes. Thus, since heavy metals are non-biodegradable, highly toxic and very common in many polluted environments, the bacterial isolates analyzed in this study, definitely hold great promises for the development of new and innovative bioremediation methodologies.

Reference:

De J, Ramaiah N, Vardanyan L (2008) Detoxification of Toxic Heavy Metals by Marine Bacteria

Highly Resistant to Mercury. Marine Biotechnol 10 (4): 471-477  

Isolation of plastic degrading deep-sea bacteria

Plastics are a serious source of marine pollution and thousands of tonnes find their way into our oceans in various forms. These can cause serious damage or death to many sea creatures such as birds and turtles, as well as introducing toxic chemicals into the wider environment and transporting these around the world. In an attempt to reduce our contribution of dangerous plastics to the environment we have manufactured biodegradable plastics such as polycaprolactone (PCL) which is used in a variety of products; however, even these substances are not thought to be totally biodegradable as the hydrocarbons are very difficult to break down biologically and instead are reduced to microplastic particles. Many of these particles find their way to the sea floor and this is the environment which takes the spotlight in this study.

In a previous paper, the authors reported discovery of PCL degrading bacteria found between 300 and 600m below sea level, however here, they aimed to investigate the presence and degrading ability of bacteria found much deeper, between 5000 and 7000m. Samples were taken from the Kurile and Japan Trenches and indeed, 13 strains of PCL degrading bacteria were found. These were isolated and characterised using PCR to amplify the 16S rRNA genes which were then sequenced. The degrading ability of the strains was determined by the formation of clear zones around the colonies when grown on PCL enriched agar. However, when tested on other plastic enriched agars, none of the strains produced clear zones (which indicate biodegradation).

By using the gene sequence analysis the authors found that the strains were related to previously described species. 8 of the strains were closely related to Moritella sp., 3 were closely related to Shewanella sp. and the final 2 were closely related to Psychrobacter sp. and Pseudomonas sp., respectively. As none of these related species have previously been reported as plastic biodegraders, this is the first discovery of PCL degrading bacteria in the deep sea, capable of withstanding the low-temperature, high-pressure conditions.

This study has a positive message as plastic biodegradation, which was relatively understudied and not well understood, is obviously possible to an extent and if we continue to use predominantly aliphatic-polyesters such as PCL which are biodegradable, we could help to reduce the effects of plastics on the environment. Furthermore, as these bacteria have been genetically sequenced, it may be that with the help of metagenomics we can identify other species with the same biodegrading capabilities in other habitats. However, the positive message should be taken with caution as the study does not take into account macro plastics which are the form that most commonly kills marine vertebrates.

A review of: Sekiguchi T, Sato T, Enoki M, Kanehiro H, Uematsu K, and Kato C (2010) Isolation and characterisation of biodegradable plastic degrading bacteria from deep-sea environments. JAMSTEC Rep. Res. Dev. 11: 33-41.

Early microbial biofilm formation on marine plastic debris

Plastic debris is a well known pollutant which is abundant worldwide, and affects all habitats. The threats of plastics in the marine environment vary from, ingestion by marine organisms, transporting bound organic pollutants such as polychlorinated biphenyls and entanglement.

Although most plastics are positively buoyant and are spread by wind, some sink below the surface and into the water column, or further into sediment.

Any surface in the marine environment will become colonised by microorganisms. Biofilm formation, leading to biofouling, develops through four discrete phases; adsorption of dissolved organic molecules, attachment of bacterial cells, attachment of unicellular eukaryotes and attachment of larvae and spores. Bacterial attachment is a well controlled, where attached cells produce extracellular polymers to create complex matrixes.

Understanding the mechanisms that determine the behaviour of plastic debris in the marine environment e.g/ buoyancy is essential for creating any possible evidence-based management programme. The investigators in this study aim to characterise early biofilm formation on plastic debris.

Plastic bags (20 x 28cm) were attached to weighted Perspex boards (22 x 26mm), and suspended 2m below the surface of the water in Queen Anne’s Battery (Plymouth, UK). Boards were sampled weekly for 3 weeks, between July and August 2010; mean water temperature was 16.2^oC, salinity 33.3. Sampled boards were kept in seawater, and analysed within an hour of removal. Plastic was removed from the boards and cut into smaller squares (10 x 10cm); loosely attached material was washed off and the plastic was cut smaller again (1 x 2cm) which were used for all subsequent analyses.

A quantitative biofilm assay was used after modification of a previous protocol, and a quantitative hydrophobicity assay was also developed with modification of an existing protocol.

Biofilm formation was apparent on the submerged plastic after 1 week and significantly increased for the remaining duration. Coinciding with biofilm formation, the plastic surface became less hydrophobic during the investigation. The plastic started by floating on the surface of the water, in contact with the air, and subsequently sank each week until below the surface with signs of neutral buoyancy.

In this study, the plastic in week three after being cleaned to remove any biofouling behaved the same as the control plastic, floating at air-seawater interface, which agreed with previous research.

The number of heterotrophic bacteria that could be cultured, found on the plastic increased during the experiment from 1.4 x 10⁴ cells cm^-2 at week one to, 1.2 x 10⁵ cells cm^-2 at week three.

A large part in tackling what has become an international marine pollution problem is establishing a foundational understanding of the ways plastic debris behaves in the environment and the factors which influence this. Concordant changes in the physiochemical properties of plastic lie with the rapid development of microbial biofilms. Although plastic is readily colonised by bacteria, there is no evidence of potential plastic-degraders during early attachment.

Lobelle, D. & Cunliffe, M., 2011. Early microbial biofilm formation on marine plastic debris. Marine Pollution Bulletin. 62: 197-200.

Salmonella: The forgotten pathogen

Bathing water quality is an important indicator of environmental quality. Surface waters and reservoirs are vulnerable to pollution due to industrial, urban and agricultural effluents discharges which often contain a mixture of non-pathogenic and pathogenic bacteria that have been shown to be the cause of several human minor morbidity diseases. To comply with the 1976 European Bathing Water Directive, 80% of samples taken during the bathing season must meet the quality standards for the faecal indicators (which are given in the paper), and Salmonella must be absent in 1 litre and enterovirus absent from 10 litres.

Salmonella spp. is a common and expansively distributed pathogenic microorganism, and is often the recorded cause of gastroenteritis in humans; with some serotypes leading to worse illnesses such as typhoid and paratyphoid fevers.

The aim of the investigation was to assess any possible associations between the pervasiveness of Salmonella spp. and the levels of indicator microorganisms established by the new EU Directive, to assess the importance for public health of the analysis of this specific pathogen in bathing waters. The authors compared the conventional method of testing to a rapid and automated immunoassay; this detects Oxygen and Hydrogen antigens, using the Enzyme Linked Fluorescent Assay method, which is able to detect motile and non-motile strains in 45 minutes after pre-enrichment (a much cheaper and less time consuming detection of Salmonella).

Water samples for analysis were taken from 62 points: 45 coastal waters, three transitional waters and 14 inland waters in the north of Portugal; and 36 monitoring points: 26 coastal waters and 10 transitional and inland waters were analysed for the concentration of indicator microorganisms, during the bathing seasons 2005-2008.

The samples were collected from approximately 30cm below the water at chest level (typically around 1.2-1.5m) and processed within 4-6 hours of collection. A total of 540 samples were analysed.

The analysis for Salmonella spp. was qualitative. The methods are given in the paper, but briefly, the cultural pre-enrichment and enrichment protocol, plating on selective agars and preliminary confirmation.

A total of 125 out of the 540 samples tested positive for the presence of Salmonella spp.; 35 serotypes of Salmonella spp. were identified; 18 recovered from sea water, 23 from rivers and six from transitional waters. The most frequent serotype in rivers was S. enteritidis found in 20%, and the most common in marine waters was S. typhimurium found in 15.6%.

Despite the improved control over bathing water quality, the results given in the paper seem to suggest that two microbiological indicators are insufficient to protect the health of bathers, as there is no unambiguous relationship between the presence of these indicators and the presence of pathogenic microorganisms in the water.

This study emphasizes the importance of public health in the assessment of bathing water quality, suggesting routine sampling should be carefully applied, defining health hazard indicators and employing strategies on a beach-to-beach basis, that strengthen public health inspection systems and information, in order to enhance health prevention.

Mansilha, C. R., Coelho, C. A., Reinas, A., Moutinho, A., Ferreira, S., Pizarro, C., & Tavares, A., 2010. Salmonella: The forgotten pathogen: Health hazards of compliance with European Bathing Water Legislation. Marine Pollution Bulletin. 60 (6): 819-826.