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1.  Microbial production of surfactants and their commercial potential. 
Many microorganisms, especially bacteria, produce biosurfactants when grown on water-immiscible substrates. Biosurfactants are more effective, selective, environmentally friendly, and stable than many synthetic surfactants. Most common biosurfactants are glycolipids in which carbohydrates are attached to a long-chain aliphatic acid, while others, like lipopeptides, lipoproteins, and heteropolysaccharides, are more complex. Rapid and reliable methods for screening and selection of biosurfactant-producing microorganisms and evaluation of their activity have been developed. Genes involved in rhamnolipid synthesis (rhlAB) and regulation (rhlI and rhlR) in Pseudomonas aeruginosa are characterized, and expression of rhlAB in heterologous hosts is discussed. Genes for surfactin production (sfp, srfA, and comA) in Bacillus spp. are also characterized. Fermentative production of biosurfactants depends primarily on the microbial strain, source of carbon and nitrogen, pH, temperature, and concentration of oxygen and metal ions. Addition of water-immiscible substrates to media and nitrogen and iron limitations in the media result in an overproduction of some biosurfactants. Other important advances are the use of water-soluble substrates and agroindustrial wastes for production, development of continuous recovery processes, and production through biotransformation. Commercialization of biosurfactants in the cosmetic, food, health care, pulp- and paper-processing, coal, ceramic, and metal industries has been proposed. However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants. Perspectives for future research and applications are also discussed.
PMCID: PMC232600  PMID: 9106364
2.  Assessing potential dietary toxicity of heavy metals in selected vegetables and food crops*  
Heavy metals, such as cadmium, copper, lead, chromium and mercury, are important environmental pollutants, particularly in areas with high anthropogenic pressure. Their presence in the atmosphere, soil and water, even in traces can cause serious problems to all organisms, and heavy metal bioaccumulation in the food chain especially can be highly dangerous to human health. Heavy metals enter the human body mainly through two routes namely: inhalation and ingestion, ingestion being the main route of exposure to these elements in human population. Heavy metals intake by human populations through food chain has been reported in many countries. Soil threshold for heavy metal toxicity is an important factor affecting soil environmental capacity of heavy metal and determines heavy metal cumulative loading limits. For soil-plant system, heavy metal toxicity threshold is the highest permissible content in the soil (total or bioavailable concentration) that does not pose any phytotoxic effects or heavy metals in the edible parts of the crops does not exceed food hygiene standards. Factors affecting the thresholds of dietary toxicity of heavy metal in soil-crop system include: soil type which includes soil pH, organic matter content, clay mineral and other soil chemical and biochemical properties; and crop species or cultivars regulated by genetic basis for heavy metal transport and accumulation in plants. In addition, the interactions of soil-plant root-microbes play important roles in regulating heavy metal movement from soil to the edible parts of crops. Agronomic practices such as fertilizer and water managements as well as crop rotation system can affect bioavailability and crop accumulation of heavy metals, thus influencing the thresholds for assessing dietary toxicity of heavy metals in the food chain. This paper reviews the phytotoxic effects and bioaccumulation of heavy metals in vegetables and food crops and assesses soil heavy metal thresholds for potential dietary toxicity.
PMCID: PMC1764924  PMID: 17173356
Heavy metals; Dietary toxicity; Vegetables; Food crops
3.  Bioremediation of multi-metal contaminated soil using biosurfactant — a novel approach 
Indian Journal of Microbiology  2008;48(1):142-146.
An unconventional nutrient medium, distillery spent wash (1:3) diluted) was used to produce di-rhamnolipid biosurfactant by Pseudomonas aeruginosa strain BS2. This research further assessed the potential of the biosurfactant as a washing agent for metal removal from multimetal contaminated soil (Cr-940 ppm; Pb-900 ppm; Cd-430 ppm; Ni-880 ppm; Cu-480 ppm). Out of the treatments of contaminated soil with tap water and rhamnolipid biosurfactant, the latter was found to be potent in mobilization of metal and decontamination of contaminated soil. Within 36 hours of leaching study, di-rhamnolipid as compared to tap water facilitated 13 folds higher removal of Cr from the heavy metal spiked soil whereas removal of Pb and Cu was 9–10 and 14 folds higher respectively. Leaching of Cd and Ni was 25 folds higher from the spiked soil. This shows that leaching behavior of biosurfactant was different for different metals. The use of wastewater for production of biosurfactant and its efficient use in metal removal make it a strong applicant for bioremediation.
PMCID: PMC3450211  PMID: 23100708
Pseudomonas aeruginosa; Rhamnolipid Biosurfactant; Distillary waste; Metals; Column studies
4.  Diversity of Arbuscular Mycorrhizal Fungus Populations in Heavy-Metal-Contaminated Soils 
High concentrations of heavy metals have been shown to adversely affect the size, diversity, and activity of microbial populations in soil. The aim of this work was to determine how the diversity of arbuscular mycorrhizal (AM) fungi is affected by the addition of sewage-amended sludge containing heavy metals in a long-term experiment. Due to the reduced number of indigenous AM fungal (AMF) propagules in the experimental soils, several host plants with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimental soils, showing consistent differences with regard to their tolerance to the presence of heavy metals. AMF ecotypes ranged from very sensitive to the presence of metals to relatively tolerant to high rates of heavy metals in soil. Total AMF spore numbers decreased with increasing amounts of heavy metals in the soil. However, species richness and diversity as measured by the Shannon-Wiener index increased in soils receiving intermediate rates of sludge contamination but decreased in soils receiving the highest rate of heavy-metal-contaminated sludge. Relative densities of most AMF species were also significantly influenced by soil treatments. Host plant species exerted a selective influence on AMF population size and diversity. We conclude based on the results of this study that size and diversity of AMF populations were modified in metal-polluted soils, even in those with metal concentrations that were below the upper limits accepted by the European Union for agricultural soils.
PMCID: PMC91085  PMID: 9925606
5.  Reclamation of tannery polluted soil through phytoremediation 
The huge volume of sludge emanating from the tannery effluent treatment plants poses a serious environmental problem. Phytoremediation is an emerging technology in which the plants are employed to reclamate the contaminated soil strewn with heavy metals (metalloids) and toxic compounds. This work focuses the impact of application of tannery sludge on biochemical properties of 6 months old tree saplings of Azadirachta indica A. Juss. (Neem), Melia azedarach Linn. (Wild Neem) and Leucaena leucocephala (Lam) de Wit (Subabool) raised over the tannery sludge in an attempt to use these plants for phytoremediation. The plants raised over the garden soil served as the control. The porosity and water holding capacity of the tannery sludge were higher. The plant growth supporting elements such as Ca, total N2, NO3 and Mg were higher in the sludge. The plants raised over the sludge were found to be dark green with increased morphometric parameters. Electrophoretic profile revealed amplification of a few polypeptides (100, 105, 49 and 55 KDa). The levels of biomolecules and the CO2 absorption increased in 6 months old plants. There was a significant uptake and transport of chromium in all the three tree species suggesting that these plants could be employed in phytoremediation of soils contaminated with heavy metals.
PMCID: PMC3550371  PMID: 23572927
Azadirachta indica (Neem); CO2 uptake; Leucaena leucocephala (Subabool); Melia azedarach (Wild Neem); metalloids; Phytoremediation; Stress biomolecules; Tannery sludge
6.  A pilot plant for removing chromium from residual water of tanneries. 
Environmental Health Perspectives  1995;103(Suppl 1):63-65.
The purpose of this study is to develop a technical process for removing trivalent chromium from tannery wastewater via precipitation. This process can be considered an alternative that avoids a remediation procedure against the metal presence in industrial wastes. This process was verified in a treatment pilot plant located in León, México handling 10 m3/day of three types of effluents. The effluent streams were separated to facilitate the elimination of pollutants from each one. The process was based on in situ treatment and recycle to reduce problems associated with transportation and confinement of contaminated sludges. Two types of treatment were carried out in the pilot plant: The physical/chemical and biological treatments. Thirty-five experiments were conducted and the studied variables were the pH, type of flocculant, and its dose. The statistical significance of chromium samples was 94.7% for its precipitation and 99.7% for recovery. The objectives established for this phase of the development were accomplished and the overall efficiencies were measured for each stage in the pilot plant. The results were: a) chromium precipitation 99.5% from wastewater stream, b) chromium recovery 99% for recycling, and c) physical/chemical treatment to eliminate grease and fat at least 85% and 65 to 70% for the biological treatment. The tanning of a hide lot (350 pieces) was accomplished using 60% treated and recycled water without affecting the product quality. The recovered chromium liquor was also used in this hide tanning. This technical procedure is also applicable for removing heavy metals in other industrial sectors as well as in reducing water consumption rates, if pertinent adjustments are implemented.
PMCID: PMC1519324  PMID: 7621802
7.  Hazardous solid waste from metallurgical industries. 
Types of land disposed residuals from selected metal smelting and refining industries are described, as are the origin and disposition of land disposed residuals from the primary copper industry as an example. Quantities of land-disposed or stored residuals, including slags, sludges, and dusts, are given per unit of metal production for most primary and secondary metal smelting and refining industries. Assessments of the hazard potential of residuals are given. Present treatment and disposal of residuals are discussed and assessed for health and environmental protection. Possible technologies for protection of ground and surface water contamination are presented. These include lined lagoons, chemical fixation of sludge, and ground sealing. Possibilities of resource recovery from residuals are discussed. Data are presented showing attenuation of heavy metal ions and fluorides in selected soils. The leachability and mobility of smelting and refining residuals constituents, including heavy metals and fluorides, and other potential toxicants in specific soil, geologic, and hydrologic disposal environments must be carefully considered in setting disposal requirements.
PMCID: PMC1637303  PMID: 738242
8.  Bioremediation of Heavy Metals in Liquid Media Through Fungi Isolated from Contaminated Sources 
Indian Journal of Microbiology  2011;51(4):482-487.
Wastewater particularly from electroplating, paint, leather, metal and tanning industries contain enormous amount of heavy metals. Microorganisms including fungi have been reported to exclude heavy metals from wastewater through bioaccumulation and biosorption at low cost and in eco-friendly way. An attempt was, therefore, made to isolate fungi from sites contaminated with heavy metals for higher tolerance and removal of heavy metals from wastewater. Seventy-six fungal isolates tolerant to heavy metals like Pb, Cd, Cr and Ni were isolated from sewage, sludge and industrial effluents containing heavy metals. Four fungi (Phanerochaete chrysosporium, Aspegillus awamori, Aspergillus flavus, Trichoderma viride) also were included in this study. The majority of the fungal isolates were able to tolerate up to 400 ppm concentration of Pb, Cd, Cr and Ni. The most heavy metal tolerant fungi were studied for removal of heavy metals from liquid media at 50 ppm concentration. Results indicated removal of substantial amount of heavy metals by some of the fungi. With respect to Pb, Cd, Cr and Ni, maximum uptake of 59.67, 16.25, 0.55, and 0.55 mg/g was observed by fungi Pb3 (Aspergillus terreus), Trichoderma viride, Cr8 (Trichoderma longibrachiatum), and isolate Ni27 (A. niger) respectively. This indicated the potential of these fungi as biosorbent for removal of heavy metals from wastewater and industrial effluents containing higher concentration of heavy metals.
PMCID: PMC3209935  PMID: 23024411
Industrial wastewater; Fungi; Biosorbent; Bioaccumulation; Heavy metals
9.  Influence of urban activity in modifying water parameters, concentration and uptake of heavy metals in Typha latifolia L. into a river that crosses an industrial city 
Heavy metals like Cu, Cd, Pb, Ni, Co and Cr can naturally be found almost all over this planet in various amounts. Urban activities such as heavy metal industry, traffic and waste can rapidly increase the metal concentrations in a fresh water ecosystem.
This study was done in natural conditions to capture as many aspects in heavy metals pollution and bioremediation of Nicolina River, Romania considered a stream model which is under anthropogenic pressure. Water, sediment and leaves samples of Typha latifolia L. were collected during October 2013 and analyzed in order to assess certain heavy metals (Cu, Cd, Pb, Ni, Co and Cr) from each sampling site using GF-HR-CS-AAS with platform. Heavy metals in significant concentrations in cattail samples were correlated with the water parameters to show the possibility to use the cattail leaves as indicators in heavy metals pollution with potential in bioremediation because they can be easily harvested in autumn and this species is spread worldwide.
The levels of metals concentrations in leaves were: Cu > Ni > Cr > Pb > Co knowing that copper is an essential element for plants. The sampling time was important to draw the river diagnosis for heavy metal pollution. The samples were collected, from river, after more than 60 days without rain same as a “human patient” prepared for blood test. Cobalt was considered the metal marker because it was an element with the lowest level of usage in the city. Compared with it only lead, cadmium and copper were used intensively in the industrial activities.
T. latifolia L. can be use as an indicator for the health of the studied stream and it was noticed that the heavy metals were not accumulated, although the metal uptake was influenced by sediments and water parameters. The alkalinity of the studied river acts as an inhibitor in the bioremediation process of cattail for cadmium and copper. Lead was uptake by leaves and the water parameters influenced it but it wasn’t concentrated enough in leaves to propose this species in lead bioremediation process for Nicolina River.
PMCID: PMC4324655
Bioremediation; Heavy metals; Environmental interactions; Metal uptake; Typha latifolia; Urban activities
10.  Retention and mitigation of metals in sediment, soil, water, and plant of a newly constructed root-channel wetland (China) from slightly polluted source water 
SpringerPlus  2014;3:326.
Constructed root-channel wetland (CRCW) is a term for pre-pond/wetland/post-pond complexes, where the wetland includes plant-bed/ditch landscape and root-channel structure. Source water out of pre-ponds flows through alternate small ditches and plant beds with root-channels via a big ditch under hydraulic regulation. Then source water flows into post-ponds to finish final polishing. This article aims to explore the potential of components of a pilot CRCW in China on mitigating metals in micro-polluted source water during its initial operation stage. We investigated six heavy metals (Cd, Cr, Cu, Ni, Zn, and Pb) in surface sediment, plant-bed subsurface soil, water, and aquatic plants during 2012–2013. Monitoring results showed that pond/ditch sediments and plant-bed soil retained a significant amount of Cr, Ni, and Zn with 93.1%, 72.4%, and 57.5% samples showing contamination factor above limit 1 respectively. Remarkably the high values of metal enrichment factor (EF) occurred in root-channel zones. Water monitoring results indicated that Ni, Zn, and Pb were removed by 78.5% (66.7%), 57.6% (59.6%), and 26.0% (7.5%) in east (west) wetland respectively. Mass balance estimation revealed that heavy metal mass in the pond/ditch sediments accounted for 63.30% and that in plant-bed soil 36.67%, while plant uptake occupied only 0.03%. The heavy metal accretion flux in sediments was 0.41 - 211.08 μg · cm-2 · a-1, less than that in plant-bed soil (0.73 - 543.94 μg · cm-2 · a-1). The 1.83 ha wetland has retained about 86.18 kg total heavy metals within 494 days after operation. This pilot case study proves that constructed root-channel wetland can reduce the potential ecological risk of purified raw water and provide a new and effective method for the removal of heavy metals from drinking water sources.
Electronic supplementary material
The online version of this article (doi:10.1186/2193-1801-3-326) contains supplementary material, which is available to authorized users.
PMCID: PMC4094763  PMID: 25032090
Pond-wetland complexes; Root-channel zone; Plant-bed/ditch system; Heavy metal; Enrichment factor; Potential ecological risk; Mass balance
11.  Interaction of Soil Heavy Metal Pollution with Industrialisation and the Landscape Pattern in Taiyuan City, China 
PLoS ONE  2014;9(9):e105798.
Many studies indicated that industrialization and urbanization caused serious soil heavy metal pollution from industrialized age. However, fewer previous studies have conducted a combined analysis of the landscape pattern, urbanization, industrialization, and heavy metal pollution. This paper was aimed at exploring the relationships of heavy metals in the soil (Pb, Cu, Ni, As, Cd, Cr, Hg, and Zn) with landscape pattern, industrialisation, urbanisation in Taiyuan city using multivariate analysis. The multivariate analysis included correlation analysis, analysis of variance (ANOVA), independent-sample T test, and principal component analysis (PCA). Geographic information system (GIS) was also applied to determine the spatial distribution of the heavy metals. The spatial distribution maps showed that the heavy metal pollution of the soil was more serious in the centre of the study area. The results of the multivariate analysis indicated that the correlations among heavy metals were significant, and industrialisation could significantly affect the concentrations of some heavy metals. Landscape diversity showed a significant negative correlation with the heavy metal concentrations. The PCA showed that a two-factor model for heavy metal pollution, industrialisation, and the landscape pattern could effectively demonstrate the relationships between these variables. The model explained 86.71% of the total variance of the data. Moreover, the first factor was mainly loaded with the comprehensive pollution index (P), and the second factor was primarily loaded with landscape diversity and dominance (H and D). An ordination of 80 samples could show the pollution pattern of all the samples. The results revealed that local industrialisation caused heavy metal pollution of the soil, but such pollution could respond negatively to the landscape pattern. The results of the study could provide a basis for agricultural, suburban, and urban planning.
PMCID: PMC4174504  PMID: 25251460
12.  Long-Term Effects of Metal-Rich Sewage Sludge Application on Soil Populations of Bradyrhizobium japonicum 
The application of sewage sludge to land may increase the concentration of heavy metals in soil. Of considerable concern is the effect of heavy metals on soil microorganisms, especially those involved in the biocycling of elements important to soil productivity. Bradyrhizobium japonicum is a soil bacterium involved in symbiotic nitrogen fixation with Glycine max, the common soybean. To examine the effect of metal-rich sludge application on B. japonicum, the MICs for Pb, Cu, Al, Fe, Ni, Zn, Cd, and Hg were determined in minimal media by using laboratory reference strains representing 11 common serogroups of B. japonicum. Marked differences were found among the B. japonicum strains for sensitivity to Cu, Cd, Zn, and Ni. Strain USDA 123 was most sensitive to these metals, whereas strain USDA 122 was most resistant. In field studies, a silt loam soil amended 11 years ago with 0, 56, or 112 Mg of digested sludge per ha was examined for total numbers of B. japonicum by using the most probable number method. Nodule isolates from soybean nodules grown on this soil were serologically typed, and their metal sensitivity was determined. The number of soybean rhizobia in the sludge-amended soils was found to increase with increasing rates of sludge. Soybean rhizobia strains from 11 serogroups were identified in the soils; however, no differences in serogroup distribution or proportion of resistant strains were found between the soils. Thus, the application of heavy metal-containing sewage sludge did not have a long-term detrimental effect on soil rhizobial numbers, nor did it result in a shift in nodule serogroup distribution.
PMCID: PMC203658  PMID: 16347281
13.  The Dublin SURGE Project: geochemical baseline for heavy metals in topsoils and spatial correlation with historical industry in Dublin, Ireland 
The Dublin SURGE (Soil Urban Geochemistry) Project is Dublin’s first baseline survey of heavy metals and persistent organic pollutants in topsoils and is part of a Europe-wide initiative to map urban geochemical baselines in ten cities. 1,058 samples were collected as part of a stratified random sampling programme in the greater Dublin area to give an overview of baseline conditions in the city. Samples were analysed for 31 inorganic elements including heavy metals. Analysis of results indicates that the concentrations of lead, copper, zinc and mercury are strongly influenced by human activities, with elevated concentrations in the city docklands, inner city and heavy industry areas. Sources of heavy metals in these areas may include historical industry, coal burning, re-use of contaminated soil, modern traffic and leaded paint and petrol. Concentrations of other inorganic elements in topsoil show patterns which are strongly related to regional bedrock parent material. The spatial distributions of heavy metals, in particular Pb and As, are explored in detail with respect to regional geology and the influence of historical industry on soil quality. Exploratory data, geostatistical and correlation analyses suggest that the concentrations of heavy metals tend to increase as the intensity of historical industrial activity increases. In particular, drinks production, power generation, oil/gas/coal, metals and textile historical industries appear to be the contamination source for several heavy metals. The data provide a geochemical baseline relevant to the protection of human health, compliance with environmental legislation, land use planning and urban regeneration.
PMCID: PMC3938859  PMID: 23990170
Urban geochemistry; Heavy metals; Soil pollution; Historical industry; Human health
14.  Assessment of Heavy Metals in Municipal Sewage Sludge: A Case Study of Limpopo Province, South Africa 
Heavy metals in high concentrations can cause health and environmental damage. Nanosilver is an emerging heavy metal which has a bright future of use in many applications. Here we report on the levels of silver and other heavy metals in municipal sewage sludge. Five towns in Limpopo province of South Africa were selected and the sludge from their wastewater treatment plants (WWTPs) was collected and analysed. The acid digested sewage sludge samples were analysed using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) methods. The concentrations of silver found were low, but significant, in the range 0.22 to 21.93 mg/kg dry mass. The highest concentration of silver was found in Louis Trichardt town with a concentration of 21.93 ± 0.38 mg/kg dry mass while the lowest was Thohoyandou with a concentration of 6.13 ± 0.12 mg/kg dry mass. A control sludge sample from a pit latrine had trace levels of silver at 0.22 ± 0.01 mg/kg dry mass. The result showed that silver was indeed present in the wastewater sewage sludge and at present there is no DWAF guideline standard. The average Cd concentration was 3.10 mg/kg dry mass for Polokwane municipality. Polokwane and Louis Trichardt municipalities exhibited high levels of Pb, in excess DWAF guidelines, in sludge at 102.83 and 171.87 mg/kg respectfully. In all the WWTPs the zinc and copper concentrations were in excess of DWAF guidelines. The presence of heavy metals in the sewage sludge in excess of DWAF guidelines presents environmental hazards should the sludge be applied as a soil ameliorant.
PMCID: PMC3986993  PMID: 24595211
heavy metals; sewage sludge; effluent; silver
15.  Effect of heavy metals on germination of seeds 
With the expansion of the world population, the environmental pollution and toxicity by chemicals raises concern. Rapid industrialization and urbanization processes has led to the incorporation of pollutants such as pesticides, petroleum products, acids and heavy metals in the natural resources like soil, water and air thus degrading not only the quality of the environment, but also affecting both plants and animals. Heavy metals including lead, nickel, cadmium, copper, cobalt, chromium and mercury are important environmental pollutants that cause toxic effects to plants; thus, lessening productivity and posing dangerous threats to the agro-ecosystems. They act as stress to plants and affect the plant physiology. In this review, we have summarized the effects of heavy metals on seeds of different plants affecting the germination process. Although reports exist on mechanisms by which the heavy metals act as stress and how plants have learnt to overcome, the future scope of this review remains in excavating the signaling mechanisms in germinating seeds in response to heavy metal stress.
PMCID: PMC3783763  PMID: 24082715
Germination; heavy metals; stress; seed
16.  Spatial patterns of heavy metals in soil under different geological structures and land uses for assessing metal enrichments 
Environmental Monitoring and Assessment  2013;185(12):9871-9888.
One hundred and thirty composite soil samples were collected from Hamedan county, Iran to characterize the spatial distribution and trace the sources of heavy metals including As, Cd, Co, Cr, Cu, Ni, Pb, V, Zn, and Fe. The multivariate gap statistical analysis was used; for interrelation of spatial patterns of pollution, the disjunctive kriging and geoenrichment factor (EFG) techniques were applied. Heavy metals and soil properties were grouped using agglomerative hierarchical clustering and gap statistic. Principal component analysis was used for identification of the source of metals in a set of data. Geostatistics was used for the geospatial data processing. Based on the comparison between the original data and background values of the ten metals, the disjunctive kriging and EFG techniques were used to quantify their geospatial patterns and assess the contamination levels of the heavy metals. The spatial distribution map combined with the statistical analysis showed that the main source of Cr, Co, Ni, Zn, Pb, and V in group A land use (agriculture, rocky, and urban) was geogenic; the origin of As, Cd, and Cu was industrial and agricultural activities (anthropogenic sources). In group B land use (rangeland and orchards), the origin of metals (Cr, Co, Ni, Zn, and V) was mainly controlled by natural factors and As, Cd, Cu, and Pb had been added by organic factors. In group C land use (water), the origin of most heavy metals is natural without anthropogenic sources. The Cd and As pollution was relatively more serious in different land use. The EFG technique used confirmed the anthropogenic influence of heavy metal pollution. All metals showed concentrations substantially higher than their background values, suggesting anthropogenic pollution.
PMCID: PMC3825593  PMID: 23797635
Heavy metals; Spatial variability; Land use patterns; Geostatistics; Multivariate statistics; Gap statistical analysis
17.  “NiCo Buster”: engineering E. coli for fast and efficient capture of cobalt and nickel 
Metal contamination is widespread and results from natural geogenic and constantly increasing anthropogenic sources (mainly mining and extraction activities, electroplating, battery and steel manufacturing or metal finishing). Consequently, there is a growing need for methods to detoxify polluted ecosystems. Industrial wastewater, surface water and ground water need to be decontaminated to alleviate the contamination of soils and sediments and, ultimately, the human food chain. In nuclear power plants, radioactive metals are produced; these metals need to be removed from effluents before they are released into the environment, not only for pollution prevention but also for waste minimization. Many physicochemical methods have been developed for metal removal from aqueous solutions, including chemical coagulation, adsorption, extraction, ion exchange and membrane separation; however, these methods are generally not metal selective. Bacteria, because they contain metal transporters, provide a potentially competitive alternative to the current use of expensive and high-volume ion-exchange resins.
The feasibility of using bacterial biofilters as efficient tools for nickel and cobalt ions specific remediation was investigated. Among the factors susceptible to genetic modification in Escherichia coli, specific efflux and sequestration systems were engineered to improve its metal sequestration abilities. Genomic suppression of the RcnA nickel (Ni) and cobalt (Co) efflux system was combined with the plasmid-controlled expression of a genetically improved version of a specific metallic transporter, NiCoT, which originates from Novosphingobium aromaticivorans. The resulting strain exhibited enhanced nickel (II) and cobalt (II) uptake, with a maximum metal ion accumulation of 6 mg/g bacterial dry weight during 10 min of treatment. A synthetic adherence operon was successfully introduced into the plasmid carrying the improved NiCoT transporter, conferring the ability to form thick biofilm structures, especially when exposed to nickel and cobalt metallic compounds.
This study demonstrates the efficient use of genetic engineering to increase metal sequestration and biofilm formation by E. coli. This method allows Co and Ni contaminants to be sequestered while spatially confining the bacteria to an abiotic support. Biofiltration of nickel (II) and cobalt (II) by immobilized cells is therefore a promising option for treating these contaminants at an industrial scale.
PMCID: PMC4124493  PMID: 25104972
Bioremediation; Cobalt; Nickel; Biofilm; Biofilter; NiCoT; Synthetic biology
18.  Effect of Drying on Heavy Metal Fraction Distribution in Rice Paddy Soil 
PLoS ONE  2014;9(5):e97327.
An understanding of how redox conditions affect soil heavy metal fractions in rice paddies is important due to its implications for heavy metal mobility and plant uptake. Rice paddy soil samples routinely undergo oxidation prior to heavy metal analysis. Fraction distribution of Cu, Pb, Ni, and Cd from paddy soil with a wide pH range was investigated. Samples were both dried according to standard protocols and also preserved under anaerobic conditions through the sampling and analysis process and heavy metals were then sequentially extracted for the exchangeable and carbonate bound fraction (acid soluble fraction), iron and manganese oxide bound fraction (reducible fraction), organic bound fraction (oxidizable fraction), and residual fraction. Fractions were affected by redox conditions across all pH ranges. Drying decreased reducible fraction of all heavy metals. Curesidual fraction, Pboxidizable fraction, Cdresidual fraction, and Niresidual fraction increased by 25%, 33%, 35%, and >60%, respectively. Pbresidual fraction, Niacid soluble fraction, and Cdoxidizable fraction decreased 33%, 25%, and 15%, respectively. Drying paddy soil prior to heavy metal analysis overestimated Pb and underestimated Cu, Ni, and Cd. In future studies, samples should be stored after injecting N2 gas to maintain the redox potential of soil prior to heavy metal analysis, and investigate the correlation between heavy metal fraction distribution under field conditions and air-dried samples.
PMCID: PMC4019631  PMID: 24823670
19.  Compacted Sewage Sludge as a Barrier for Tailings: The Heavy Metal Speciation and Total Organic Carbon Content in the Compacted Sludge Specimen 
PLoS ONE  2014;9(6):e100932.
Acid mine drainage (AMD) was the main environmental problem facing the mining industry. For AMD had high heavy metals content and low pH, the compacted sewage sludge might be a barrier for tailings whose oxidation and weathering produced AMD, with its own carbon source, microorganism reduction ability and impermeability. To study the heavy metals environmental risk, under the simulate AMD, the deionized water (DW), and the pH 2.1 sulfuric acid water (SA) seepage conditions, respectively, the changes of the chemical speciation of heavy metals Cd, Cu, Fe, Ni, Zn and total organic carbon (TOC) content in the compacted sewage sludge were assessed in the different periods. The results indicated according to the distribution of heavy metals, the potential mobility was for Cd: 6.08 under AMD, 7.48 under SA, ∞ under DW; for Cu: 0.08 under AMD, 0.17 under SA, 0.59 under DW; for Fe: 0.15 under AMD, 0.22 under SA, 0.22 under DW; for Ni: 2.60 under AMD, 1.69 under SA, 1.67 under DW; and for Zn: 0.15 under AMD, 0.23 under SA and 0.21 under DW at the second checking time. TOC content firstly decreased from 67.62±0% to 66.29±0.35%, then increased to 67.74±0.65% under the AMD seepage while TOC decreased to 63.30±0.53%, then to 61.33±0.37% under the DW seepage, decreased to 63.86±0.41%, then to 63.28±0.49% under SA seepage. That indicated under the AMD seepage, the suitable microorganisms communities in the compacted sewage sludge were activated. And the heavy metals environmental risk of compacted sewage sludge was lower with AMD condition than with other two. So the compacted sewage sludge as a barrier for tailings was feasible as the aspect of environmental risk assessment.
PMCID: PMC4076238  PMID: 24979755
20.  Multivariate statistical assessment of heavy metal pollution sources of groundwater around a lead and zinc plant 
The contamination of groundwater by heavy metal ions around a lead and zinc plant has been studied. As a case study groundwater contamination in Bonab Industrial Estate (Zanjan-Iran) for iron, cobalt, nickel, copper, zinc, cadmium and lead content was investigated using differential pulse polarography (DPP). Although, cobalt, copper and zinc were found correspondingly in 47.8%, 100.0%, and 100.0% of the samples, they did not contain these metals above their maximum contaminant levels (MCLs). Cadmium was detected in 65.2% of the samples and 17.4% of them were polluted by this metal. All samples contained detectable levels of lead and iron with 8.7% and 13.0% of the samples higher than their MCLs. Nickel was also found in 78.3% of the samples, out of which 8.7% were polluted. In general, the results revealed the contamination of groundwater sources in the studied zone. The higher health risks are related to lead, nickel, and cadmium ions. Multivariate statistical techniques were applied for interpreting the experimental data and giving a description for the sources. The data analysis showed correlations and similarities between investigated heavy metals and helps to classify these ion groups. Cluster analysis identified five clusters among the studied heavy metals. Cluster 1 consisted of Pb, Cu, and cluster 3 included Cd, Fe; also each of the elements Zn, Co and Ni was located in groups with single member. The same results were obtained by factor analysis. Statistical investigations revealed that anthropogenic factors and notably lead and zinc plant and pedo-geochemical pollution sources are influencing water quality in the studied area.
PMCID: PMC3561054  PMID: 23369182
Heavy metals; Groundwater; Differential pulse polarography; Multivariate data analysis
21.  Trace Elements Analysis in Forage Samples from a US Navy Bombing Range (Vieques, Puerto Rico) 
Plants are good environmental sensors of the soil conditions in which they are growing. They also respond directly to the state of air. The tops of plants are collectors of air pollutants, and their chemical composition may be a good indicator for contaminated-areas when it is assessed against background values obtained for unpolluted vegetation. Both, aquatic and terrestrial plants are known to bioaccumulate heavy metals and therefore represent a potential source of these contaminants to the human food chain. An evaluation of heavy metals was conducted from vegetation samples collected at the Atlantic Fleet Weapons Training Facilities (AFWTF) in Vieques, Puerto Rico. In order to understand the potential risks associated to heavy metal mobilization through biological systems, it is first necessary to establish background values obtained from reference locations. This information allows a better interpretation of the significance of anthropogenic factors in changing trace elements status in soil and plants. Since Guánica State Forest is located at a similar geoclimatic zone as the AFWTF, samples at this site were used as a standard reference material and as experimental controls. Both sampling and analysis were conducted as previously described in standardized protocols using acid digestion of dry ashes. Then, levels of heavy metals were obtained by air-acetylene flame detection in an atomic absorption spectrophotometer. Our results from the samples taken at the AFWTF indicate mobilization of undesirable trace elements through the marine and terrestrial food web. Since plants naturally remove heavy metals from soils, they could be employed for the restoration of this and similarly contaminated sites.
PMCID: PMC3810629  PMID: 16705826
Vieques; trace elements; military wastes; phytoremediation; tropics; bioindicators
22.  Exogenous Isolation of Mobilizing Plasmids from Polluted Soils and Sludges 
Exogenous plasmid isolation was used to assess the presence of mobilizing plasmids in several soils and activated sludges. Triparental matings were performed with Escherichia coli (a member of the γ subgroup of the Proteobacteria) as the donor of an IncQ plasmid (pMOL155, containing the heavy metal resistance genes czc: Cor, Znr, and Cdr), Alcaligenes eutrophus (a member of the β subgroup of the Proteobacteria) as the recipient, and indigenous microorganisms from soil and sludge samples as helper strains. We developed an assay to assess the plasmid mobilization potential of a soil ecosystem on the basis of the number of transconjugants obtained after exogenous isolations. After inoculation into soil of several concentrations of a helper strain (E. coli CM120 harboring IncP [IncP1] mobilizing plasmid RP4), the log numbers of transconjugants obtained from exogenous isolations with different soil samples were a linear function of the log numbers of helper strain CM120(RP4) present in the soils. Four soils were analyzed for the presence of mobilizing elements, and mobilizing plasmids were isolated from two of these soils. Several sludge samples from different wastewater treatment plants yielded much higher numbers of transconjugants than the soil samples, indicating that higher numbers of mobilizing strains were present. The mobilizing plasmids isolated from Gent-O sludge and one plasmid isolated from Eislingen soil hybridized to the repP probe, whereas the plasmids isolated from Essen soil did not hybridize to a large number of rep probes (repFIC, repHI1, repH12, repL/M, repN, repP, repT, repU, repW, repX). This indicates that in Essen soil, broad-host-range mobilizing plasmids belonging to other incompatibility groups may be present.
PMCID: PMC201399  PMID: 16349216
23.  Soil and plant factors influencing the accumulation of heavy metals by plants. 
The use of plants to monitor heavy metal pollution in the terrestrial environment must be based on a cognizance of the complicated, integrated effects of pollutant source and soil-plant variables. To be detectable in plants, pollutant sources must significantly increase the plant available metal concentration in soil. The major factor governing metal availability to plants in soils is the solubility of the metal associated with the solid phase, since in order for root uptake to occur, a soluble species must exist adjacent to the root membrane for some finite period. The rate of release and form of this soluble species will have a strong influence on the rate and extent of uptake and, perhaps, mobility and toxicity in the plant and consuming animals. The factors influencing solubility and form of available metal species in soil vary widely geographically and include the concentration and chemical form of the element entering soil, soil properties (endogenous metal concentration, mineralogy, particle size distribution), and soil processes (e.g., mineral weathering, microbial activity), as these influence the kinetics of sorption reactions, metal concentration in solution and the form of soluble and insoluble chemical species. The plant root represents the first barrier to the selective accumulation of ions present in soil solution. Uptake and kinetic data for nutrient ions and chemically related nonnutrient analogs suggest that metabolic processes associated with root absorption of nutrients regulate both the affinity and rate of absorption of specific nonnutrient ions. Detailed kinetic studies of Ni, Cd, and Tl uptake by intact plants demonstrate multiphasic root absorption processes over a broad concentration range, and the use of transport mechanisms in place for the nutrient ions Cu, Zn, and K. Advantages and limitations of higher plants as indicators of increased levels of metal pollution are discussed in terms of these soil and plant phenomena.
PMCID: PMC1637297  PMID: 367766
24.  Effect of Metal-Rich Sludge Amendments on the Soil Microbial Community 
The effects of heavy-metal-containing sewage sludge on the soil microbial community were studied in two agricultural soils of different textures, which had been contaminated separately with three predominantly single metals (Cu, Zn, and Ni) at two different levels more than 20 years ago. We compared three community-based microbiological measurements, namely, phospholipid fatty acid (PLFA) analysis to reveal changes in species composition, the Biolog system to indicate metabolic fingerprints of microbial communities, and the thymidine incorporation technique to measure bacterial community tolerance. In the Luddington soil, bacterial community tolerance increased in all metal treatments compared to an unpolluted-sludge-treated control soil. Community tolerance to specific metals increased the most when the same metal was added to the soil; for example, tolerance to Cu increased most in Cu-polluted treatments. A dose-response effect was also evident. There were also indications of cotolerance to metals whose concentration had not been elevated by the sludge treatment. The PLFA pattern changed in all metal treatments, but the interpretation was complicated by the soil moisture content, which also affected the results. The Biolog measurements indicated similar effects of metals and moisture to the PLFA measurements, but due to high variation between replicates, no significant differences compared to the uncontaminated control were found. In the Lee Valley soil, significant increases in community tolerance were found for the high levels of Cu and Zn, while the PLFA pattern was significantly altered for the soils with high levels of Cu, Ni, and Zn. No effects on the Biolog measurements were found in this soil.
PMCID: PMC124700  PMID: 16349483
25.  Distribution of Biosurfactant-Producing Bacteria in Undisturbed and Contaminated Arid Southwestern Soils 
Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. However, little is known about the distribution of biosurfactant-producing bacteria in the environment. The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites. A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R2A agar. The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose. Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested. The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates. Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a Flavobacterium sp. Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates). Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions. These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates. In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay. Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils.
PMCID: PMC161513  PMID: 12788727

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