Applications of Bioremediation and Phytoremediation.

This article has been published in the journal Science Progress, which I am an Editor of. You can download a free pdf version, with figures etc. from this link: http://stl.publisher.ingentaconnect.com/content/stl/sciprg/2013/00000096/00000004/art00004

Introduction.

The decontamination of soil and water from pollutants using microorganisms (bioremediators) is known as bioremediation¹. There are essentially two approaches, described as in situ and ex situ. In situ methods are those in which the contaminated material is treated at the site, whereas when the material is physically removed to be treated elsewhere it is referred to as ex situ. Some technologies that are related to bioremediation include those of phytoremediation^2,3, and are outlined below. It is possible for bioremediation to occur under natural conditions, or it can be stimulated, e.g. by the application of fertilizers (biostimulation), and more recently it has been shown that through the addition of matched microbe strains to the medium, the effectiveness of the resident microbe population to decompose contaminants may be enhanced. It should not be imagined that every type of contaminant can be disposed of by means of this approach, and e.g. heavy metal contaminants, such as Cd²⁺ and Pb²⁺, tend to resist interception by microorganisms, which can at best change their oxidation state so that the solubility of the cations is decreased. The better way is to physically remove heavy metals using phytoremediation, by which the toxins are bioaccumulated into the body of plants, above ground, which can then be harvested and removed. By measuring the Oxidation Reduction Potential (redox) in soil and groundwater, along with pH, temperature, O₂ tension, concentrations of electron acceptors and donors, and of decomposition products, such as CO₂, a measure of the bioremediation process can be obtained. Table 1 shows different biological decomposition processes, the rates of which decrease in decreasing order of the redox potential (fastest at higher potentials), although the detail of the overall bioremediation process per se is only scantly indicated by such values. To gain insight over a larger area, sufficient measurements should be made on and around the contaminated site such that contours of equal redox potential can be drawn. It is further necessary to perform analyses to ascertain that the levels of the contaminating compounds (and their products of decomposition) are below regulatory limits.

 

Table 1⁴. Biodegradation processes of decreasing rate according to decreasing redox potential.

Process	Reaction	Redox potential (Eh in mV)
Aerobic:	O2 + 4e− + 4H+ → 2H2O	600 ~ 400
Anaerobic:
Denitrification	2NO3− + 10e− + 12H+ → N2 + 6H2O	500 ~ 200
Manganese(IV) reduction	MnO2 + 2e− + 4H+ → Mn2+ + 2H2O	400 ~ 200
Iron(III) reduction	Fe(OH)3 + e− + 3H+ → Fe2+ + 3H2O	300 ~ 100
Sulfate reduction	SO42− + 8e− +10 H+ → H2S + 4H2O	0 ~ −150
Fermentation	2CH2O → CO2 + CH4	−150 ~ −220

Bioremediation can be used in locations that cannot readily be treated other than by excavation, e.g. spillages of petrol or chlorinated solvents which may contaminate groundwater. This is usually a much cheaper approach than excavating material to be disposed of elsewhere, or through other ex situ strategies, and which reduces or eliminates the need for "pump and treat", which is often employed where clean groundwater has been contaminated. The process may be enhanced by the addition of appropriate oxidising or reducing amendment agents. There is scope too for the creation of genetically modified microorganisms that are specifically tailored for bioremediation⁵, e.g. the most radioresistent organism known so far, the aptly named bacterium Deinococcus radiodurans has been modified to consume and digest toluene and mercury cations in the presence of high level nuclear waste⁶.

Some applications of microbial biodegradation.

The move toward finding “green” ways to ameliorate many environmental woes, including dealing with polluted environments, has led to a rising focus toward microbial degradation. Such methods of bioremediation and biotransformation exploit the remarkable diversity of xenobiotic metabolism by microbes. Thus, an enormous range of polluting materials may be addressed, including hydrocarbons (e.g. from oil-spills), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), heterocyclics, pharmaceutical substances, pesticides, heavy metals (e.g. Cd²⁺, Pb²⁺, Cu²⁺, Zn²⁺) and various radionuclides (e.g. Cs⁺, Sr²⁺). Detailed genomic, metagenomic, proteomic, bioinformatic and other high-throughput analytical techniques, as applied to environmentally important microorganisms, have disclosed key features of critical biodegradative pathways and the ability of such organisms to adapt to changing environmental conditions and stress factors. To achieve a truly sustainable society, it is mandatory to reduce the environmental impact of humans, and achieving this through the use of the remarkable catabolic versatility of microorganisms to degrade or convert a variety of polluting compounds is a potential “holy grail”. Through genome-based global studies, unparalleled advances are now possible from in silico (meaning, "performed on a computer, or by computer simulation") views of metabolic and regulatory networks, along with providing insight into the evolution of degradation pathways and of molecular adaptation strategies in microorganisms, at the behest of changing environmental conditions. The degradation of PAHs provides a good example of the use of these methods⁷.

While it was formerly considered that the mineralization of aromatic hydrocarbons and halogenated compounds is probably not possible in the absence of oxygen, the more recent discovery of previously unknown anaerobic hydrocarbon-degrading and reductively dehalogenating bacteria has shown that these processes do indeed occur in nature. Through an increasing application of genomics in the field of environmental microbiology, novel molecular insights into these new metabolic properties are now possible. The ~4.7 Mb genome of the facultative denitrifying Aromatoleum aromaticum strain EbN1 was the first to be determined for an anaerobic hydrocarbon degrader. The genome sequence revealed about two dozen gene clusters, which included a number of paralogs, in a coding for a complex catabolic network for anaerobic and aerobic degradation of aromatic compounds. Genomes of anaerobic hydrocarbon degrading bacteria were recently sequenced for the iron-reducing species Geobacter metallireducens (accession nr. NC_007517) and the perchlorate-reducing Dechloromonas aromatica (accession nr. NC_007298). Complete genome sequences also determined for bacteria capable of anaerobic degradation of halogenated hydrocarbons by halorespiration are: the ~1.4 Mb genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1 and the ~5.7 Mb genome of Desulfitobacterium hafniense strain Y51. All these bacteria were shown⁸ to contain multiple paralogous genes for reductive dehalogenases, while unprecedented insights were attained into the evolution of reductive dehalogenation and differing strategies for niche adaptation. Previously, it was demonstrated that Desulfitobacterium chlororespirans – originally evaluated for halorespiration on chlorophenols – can also use some brominated compounds, such as the herbicide bromoxynil and its major metabolite as electron acceptors for growth⁹. De-iodination is possible too, although the overall reductive mechanism may differ in detail⁹.

A complete description of Desulfitobacterium hafniense strain PCP-1 has been described, which can convert pentachlorophenol (PCP) into 3-chlorophenol, dehalogenate a number of related chloroaromatic compounds and convert tetrachloroethene to trichloroethene. Four gene loci, encoding putative chlorophenol-reductive dehalogenases (CprA2 to CprA5) were detected, and the products of two of these loci have been demonstrated to dechlorinate different chlorinated phenols. The strain PCP-1 was used at the laboratory scale to degrade PCP, as present in contaminated environments, and it is concluded therefore that Desulfitobacterium hafniense PCP-1 is an excellent candidate to exploit in the development processes for the bioremediation of organohalide compounds¹⁰. A report has appeared of the isolation of the ability of the Acetobacterium sp. Strain AG, to reductively debrominate technical mixtures of Octabrominated and Pentabrominated diphenyl ethers¹¹. It should be noted that a critical factor in effective microbial degradation is the amount of the pollutant that is accessible to microorganisms. As an early example, O'Loughlin et al. showed¹² that most soil clays and cation-exchange resins accelerated the rate of biodegradation of 2-picoline by Arthrobacter sp. strain R1, as a result of adsorption of the substrate onto the clays, an apparent exception being kaolin. The directed movement of motile organisms towards or away from chemicals in the environment (chemotaxis) is an important physiological response that may contribute to effective catabolism of molecules in the environment. In addition, mechanisms for the intracellular accumulation of aromatic molecules via various transport mechanisms are also important¹³.

Biodegradation of petroleum and its products.

The ecological toxicity of petroleum is well known, for which seabirds floundering in a thick black soup of crude oil are the “poster child”.  Such marine environments are especially vulnerable to oil spills since coastal regions and the open sea are not readily isolated, and for example, the implementation of boom structures, intended to confine the oil, is of limited efficacy. In addition to such human-induced environmental catastrophes as the Deepwater Horizon disaster in the Gulf of Mexico, a remarkable 250 million litres of petroleum enter the marine environment every year from natural seepages. Oil spills are almost never as long-lived as they might at first appear, however, and the environment cleans them up substantially, given enough time. Recently, some of the agents for this beneficial activity have been identified: the hydrocarbonoclastic bacteria (HCB)¹⁴, of which Alcanivorax borkumensis was the first to be completely genome-sequenced. It would appear that other components of petroleum, including heterocyclic compounds, e.g. pyridine- and quinoline- derivatives, are degraded by similar though separate mechanisms as pertain for hydrocarbons.

Optimisation of bioreactors for waste treatment.

The human species generates colossal quantities of waste, which needs to be processed to protect the environment from it. Hence, in deriving a sustainable development programme, the use of living organisms provides a “green” alternative to chemical/engineering solutions which are costly and potentially environmentally damaging in their own right. Bioreactors provide a highly controlled “contained” space in which biotreatment processes can be carried out, in the avoidance of some of the limitations of more “open” systems. Such is the versatility of these devices that a wide range of wastes can be treated under optimized conditions. However, it is necessary to consider the genomic aspects¹⁵ of the various microorganisms involved along with their expressed transcripts and proteins. Although this is laborious using conventional genomic approaches, an adaptation of high-throughput methods of analysis – originally developed for medical applications - is now available with which to evaluate biotreatment in confined environments.

Mycoremediation¹⁶ (bioremediation by fungi).

Fungi are an essential component of the soil food web, and provide nourishment for the other biota that live in the soil. In the natural ecosystem, a realm of organisms from different kingdoms make their assault on those different substrates that are present. The rate of degradation becomes maximal when there is a good supply of nutrients present, e.g. N, P, K and other essential inorganic elements. Aspergillus and other moulds are highly efficient in decomposing starches, hemicelluloses, celluloses, pectins and other sugar polymers, and some aspergilli can degrade such intractable substrates as fats, oils, chitin, and keratin. Substrates of human origin, such as paper and textiles (cotton, jute and linen) are readily degraded by these moulds, when the process is often referred to as biodeterioration. In 1969, when the Italian city of Florence (Firenza) flooded, it was found that 74% of the isolates from a damaged Ghirlandaio fresco in the Ognissanti church were Aspergillus versicolor. Fungi function through the mycelium, which exudes extracellular enzymes and acids able to decompose lignin and cellulose, the two essential components of plant fibre. In mycoremediation the correct fungal species must be selected to target a particular pollutant, and it is possible thus to degrade successfully the nerve gases VX and sarin. By inocculating a plot of soil contaminated by diesel oil, with mycelia from oyster mushrooms, it was found that after 4 weeks, 95% of many of the PAHs had been converted to non-toxic compounds. It seems that the naturally present community of microbes acts in concert with the fungi to decompose the contaminants, finally to CO₂ plus H₂O (full mineralisation). In 2007, a cargo ship spilled 58,000 gallons of fuel along the San Francisco shoreline. Hair mats, resembling S.O.S. pads the size of a doormat, were used as sponges to soak up spilled oil. They were then collected and layered with oyster mushroom and straw. The mushrooms broke down the oil and in several weeks the resulting soil was good enough to be used to for roadside landscaping. Wood-degrading fungi are extremely effective in decomposing toxic aromatic pollutants from petroleum and also chlorinated persistent pesticides. Mycofiltration is a similar procedure, in which mycelia are used as a filter to remove toxic materials and microorganisms from water in the soil. A major protagonist of mycoremediation is Paul Stamets, who proposes¹⁷ there should be “Mycological Response Teams”, who would employ the approach to recycle and rebuild healthy soil in the area following any incident. 

Phytoremediation.

This may be defined as the treatment of environmental problems by using plants in situ so to avoid the need to excavate the contaminant material for disposal elsewhere. Phytoremediation^18,19 may be applied to the amelioration of contaminated soils, water, or air, using plants that can contain, degrade, or eliminate metals, pesticides, solvents, explosives, crude oil and its derivatives (refined fuels), and related contaminating materials. Phytoremediation has been used successfully for the restoration of abandoned metal-mine workings, and cleaning up sites where polychlorinated biphenyls have been dumped during manufacture, and for the mitigation of on-going coal mine discharges. Phytoremediation uses the natural ability of particular plants (“hyperaccumulators”, described below) to bioaccumulate, degrade, or otherwise reduce the environmental impact of contaminants in soils, water, or air. Those contaminants that have been successfully mitigated in phytoremediation projects worldwide are metals, pesticides, solvents, explosives, and crude oil and its derivatives, and the technology has become increasingly popular and has been employed at sites with soils contaminated with lead, uranium, and arsenic. A major disadvantage of phytoremediation is that it takes a relatively long time to achieve, because the process rests upon the ability of a plant to thrive in an environment that is not ideal for normal plant growth.

Advantages and limitations of phytoremediation

• Advantages:
• In terms of cost, phytoremediation is lower than that of traditional processes both in situ and ex situ.
• The plants can be easily monitored.
• There is the possibility of the recovery and re-use of valuable metals (by companies specializing in “phyto-mining”).
• It is potentially the least harmful method because it uses naturally occurring organisms and preserves the environment in a more natural state.
• Trees may be used in phytoremediation, since they grow on land of marginal quality, have long life-spans and a high flood tolerance. Willows and poplars are most commonly used, and can grow 6-8 feet (ca 2 metres) per year. For deep contamination, hybrid poplars with roots extending 30 feet deep have been used, which penetrate microscopically sized pores in the soil matrix and each tree can cycle 100 L of water per day, functioning almost as a solar powered and self-contained pump and treatment system.
• Phytoscreening is possible, in which plants may be used as biosensors for particular types of contaminants, thus giving a signal of underlying contaminant plumes, e.g. trichloroethene has been detected in the trunks of trees.
• Genetic engineering may confer improvements to phytoremediation, e.g. genes encoding a nitroreductase from a bacterium, when inserted into tobacco, increased the resistance of the plant to the toxic effects of TNT and the uptake of the material. Plants may be genetically modified to grow in soils even when the pollution levels in the soil are lethal for non-treated plants, and to absorb a greater concentration of the contaminant.

• Limitations:
• Phytoremediation is limited to the surface area and depth occupied by the plant roots.
• Slow growth and low biomass require a long-term commitment.
• Using plants, it is not possible to prevent entirely the leaching of contaminants into the groundwater (without the complete removal of the contaminated ground, which in itself does not resolve the problem of contamination).
• The survival of the plants is affected by the toxicity of the contaminated land and the general condition of the soil.
• Bio-accumulation of contaminants, especially metals, into the plants which then pass into the food chain, from primary level consumers upwards, or requires the safe disposal of the affected plant material, i.e. the plants might be eaten by animals.
• The procedure is slow.

Hyperaccumulators and biotic interactions.

If a plant is able to concentrate a particular contaminant, to a given minimum concentration (> 1000 mg/kg of dry weight for nickel, copper, cobalt, chromium or lead; or > 10,000 mg/kg for zinc or manganese), it is categorized as a hyperaccumulator. This capacity for accumulation is a result of genetic adaptation over many generations in hostile environments. Metal hyperaccumulation can affect various different factors, such as protection, interferences between different species of plants, mutualism (e.g. mycorrhizae, pollen and seed dispersal), commensalism, and biofilm.

Different possible phytoremediation methods.

Various processes that are mediated by plants or algae might be used to address environmental problems:

• Phytoextraction — uptake and concentration of substances from the environment into the plant biomass.
• Phytostabilization — reducing the mobility of substances in the environment, for example, by limiting the leaching of substances from the soil.
• Phytotransformation — chemical modification of environmental substances as a direct result of plant metabolism, often resulting in their inactivation, degradation (phytodegradation), or immobilization (phytostabilization).
• Phytostimulation — enhancement of soil microbial activity for the degradation of contaminants, typically by organisms that associate with roots. This process is also known as rhizosphere degradation. Phytostimulation can also involve aquatic plants supporting active populations of microbial degraders, as in the stimulation of atrazine degradation by hornwort.
• Phytovolatilization — removal of substances from soil or water with release into the air, sometimes as a result of phytotransformation to more volatile and/or less polluting substances.
• Rhizofiltration — filtering water through a mass of roots to remove toxic substances or excess nutrients. The pollutants remain absorbed in or adsorbed to the roots.

Phytoextraction.
 

In phytoextraction (or phytoaccumulation) plants or algae are used to extract contaminants from soils, sediments or water into harvestable plant biomass (those organisms that take larger-than-normal amounts of contaminants from the soil are called hyperaccumulators). Phytoextraction has been used more often for extracting heavy metals than for organic contaminants. The plants absorb contaminants through the root system which they then contain in the root biomass and/or move them into the stems and/or leaves. A living plant may continue to absorb contaminants until it is harvested. After harvest, a lower level of the contaminant will remain in the soil, so the growth/harvest cycle must usually be repeated through several crops to achieve a significant cleanup. The process can be repeated to affect further decontamination. There are two forms of phytoextraction:

• Natural hyper-accumulation, where plants take up the contaminants in soil unassisted.
• Induced (assisted) hyper-accumulation, in which a conditioning fluid containing a chelator or another agent is added to soil to increase metal solubility or mobilization so that the plants can absorb them more easily. In many cases natural hyperaccumulators are metallophyte plants that can tolerate and incorporate high levels of toxic metals.

Examples of phytoextraction:

• Arsenic, using the Sunflower (Helianthus annuus), or the Chinese Brake fern (Pteris vittata), a hyperaccumulator. Chinese Brake fern stores arsenic in its leaves.
• Cadmium, using willow (Salix viminalis): willow has a significant potential as a phytoextractor of cadmium (Cd), zinc (Zn), and copper (Cu), as willow has some specific characteristics like high transport capacity of heavy metals from root to shoot and huge amount of biomass production; can be used also for production of bioenergy in the biomass energy power plant.
• Cadmium and zinc, using Alpine pennycress (Thlaspi caerulescens), a hyperaccumulator of these metals at levels that would be toxic to many plants, although its growth appears to be inhibited by copper.

• Lead, using Indian Mustard (Brassica juncea), Ragweed (Ambrosia artemisiifolia), Hemp Dogbane (Apocynum cannabinum), or Poplar trees, which sequester lead in their biomass.
• Salt-tolerant (moderately halophytic) barley and/or sugar beets are commonly used for the extraction of sodium chloride (common salt) to reclaim fields that were previously flooded by sea water.
• ¹³⁷Cs and ⁹⁰Sr contaminating a pond were removed using sunflowers, following the 1986 Chernobyl accident.
• Mercury, selenium and organic pollutants including polychlorinated biphenyls (PCBs) have been removed from soils by transgenic plants containing genes for bacterial enzymes.
• Recovery of phosphate from wastewaters by algae.

Phytostabilization.

In phytostabilization the intention is to stabilize, or contain the pollutant over the long-term. There may be a number of contributing factors to this, e.g. the reduction of wind (soil) erosion by the body of the plant, but the roots of the plant can resist water (soil) erosion, immobilize the pollutants by adsorption or accumulation, and provide a zone around the roots where the pollutant can be deposited in an immobilized form. In contrast with phytoextraction, phytostabilization aims mainly to sequester pollutants in soil around the roots but not in the plant tissues. Hence the pollutants are increasingly less bioavailable, such that exposure to livestock, wildlife, and humans is reduced. Mine tailings may be stabilized by growing a vegetative cap. 

Phytotransformation.

Some plants, e.g. cannas, are able to detoxify organic pollutants - pesticides, explosives, solvents, industrial chemicals, and other xenobiotic substances  - by metabolising them. The metabolic functions of microorganisms living in association with plant roots may also metabolize these substances, as present in soil or water. Due to the complex and recalcitrant nature of many of these compounds, they cannot be broken down entirely (mineralised) to basic molecules (H₂O, CO₂, etc.) by plants and hence the term phytotransformation represents molecular alterations rather than the complete decomposition of the compound. Phytotransformation may be viewed¹⁸ as a "Green Liver" because plants behave analogously to the human liver in processing these xenobiotic compounds, introducing polar groups such as –OH to them. This is known as Phase I metabolism, similar to the way that the human liver increases the polarity of drugs and foreign compounds. In plants, it is enzymes such as nitroreductases which carry out these transformations, whereas in the human liver it is enzymes such as the Cytochrome P450s that perform the task. Phase II metabolism in the second step in phytotransformation, in which the polarity of the xenobiotic molecule is increased by combination with plant biomolecules such as glucose and amino-acids. This is called “conjugation”, and is once more similar to processes such as glucoronidation (addition of glucose) and glutathione addition reactions, catalysed by appropriate enzymes. The effect of the two metabolic steps may serve to detoxify the xenobiotic and aid its mobilization via aqueous channels. In Phase III metabolism, the xenobiotic becomes sequestered, by incorporation in a complex “lignin-type” structure, where it is kept apart from the normal functioning of the plant. The phytotransformation of trinitrotoluene (TNT) has been well studied, and a detailed mechanism proposed for it.

Phytostimulation and rhizoremediation.

This term identifies the process where compounds released from plant roots enhance microbial activity in the rhizosphere, which is the narrow region of soil around the roots of plants, and associated soil microorganisms. Soil which is not part of the rhizosphere is known as bulk soil. In rhizoremediation, microorganisms degrade soil contaminants in the rhizosphere. It is usual that those soil pollutants which are remediated by this method are highly hydrophobic organic xenobiotics that are hence unable to enter the plant. Rather than the plant being a main protagonist in this process, it creates a haven in which microorganisms in the rhizosphere are able to perform the degradation. The plant acts as a solar-powered pump, which draws in both water and the xenobiotic agent, simultaneously producing substrates (e.g. root exudates and root turnover) that assist the growth of the microbes which act as pollutant degrading agents. Microbial activity is stimulated in the rhizosphere through a number of different routes: (i) exudates, e.g. sugars, carbohydrates, amino acids, acetates, and enzymes, nourish indigenous microbe populations; (ii) root systems bring oxygen into the rhizosphere, meaning that aerobic transformations are supported; (3) the available organic carbon is enhanced through the growth of fine-root biomass; (4) mycorrhizae fungi, which are an essential component of the rhizosphere, provide unique enzymatic pathways lending the capacity to degrade pollutant molecules that would not be degraded by bacteria alone; and (5) the presence of plants (and their roots) creates a domain for microbial populations, which are activated in the rhizosphere. There have been five enzyme systems identified in soils: (i) dehalogenase (which acts in dechlorination reactions of chlorinated hydrocarbons); (ii) nitroreductase (essential for the initial step of nitroaromatic degradation); (iii) peroxidase (a critical catalyst for oxidation reactions); (iv) laccase (able to begin the decomposition of otherwise robust aromatic ring structures); (v) nitrilase (another key factor in oxidation processes). The method is limited in that when there are high concentrations of pollutants present, the plants may be overwhelmed and die. The successful use of phytostimulation has been demonstrated in the remediation of chlorinated solvents from groundwater, petroleum hydrocarbons from soil and groundwater and PAHs from soil.

Phytovolatilization.

Probably, this is the most controversial of the phytoremediation technologies, since it involves the release of contaminants either directly, or in a metabolically modified form, into the atmosphere. Phytovolatilization²⁰ has been used principally for the removal of Hg²⁺ ions which are transformed into less toxic elemental mercury²¹. Tritium (³H), a radioactive isotope of hydrogen with a half-life of about 12 years, decaying to helium, has also been removed by phytovolatilization²². A good deal more research is necessary before this strategy becomes mainstream, since there are various negative features to be addressed. For example, mercury that is released into the atmosphere from plants is likely to be recycled by precipitation and thus returned the ecosystem, and the method is restricted both to sites where the concentration of contaminants is toward the low side, and where the contamination is no deeper than the roots of the plants being used. 

Rhizofiltration.

Rhizofiltration²³ involves filtering contaminated water through a mass of roots for the extraction of contaminants, or excess nutrients, e.g. phosphorus. The contaminated water can either be collected from a waste site and taken to where plants are being hydroponically cultivated, or the plants may be planted in the area directly. In both cases, the roots draw up the water and its associated contaminants. This process is very similar to phytoextraction in that the contaminants become sequestered in the form of harvestable plant biomass. Then new plants are grown and harvested until a satisfactory degree of decontamination is achieved. It is the concentration and precipitation of heavy metals that is sought principally. While noting these similarities, the fundamental difference between the two approaches is that rhizofiltration is used in aquatic environments, while phytoextraction is applied to the decontamination of soils. There are limitations to rhizofiltration. As usual in phytoremediation methods, any contaminant that is below the rooting depth will not be extracted, and if the level of contamination is too high the plants will not grow. Depending on the type of plant and contaminant, the process may need to be continued over a protracted period, before regulatory levels are achieved. It is generally true that many different kinds of contaminants will be present – in some cases a mixture of organics and heavy metals – and thus the use of rhizofiltration alone is unlikely to succeed. Importantly, the plants chosen should be non-fodder crop to minimize poisoning animals, which might eat them in contaminated form.  That noted, the effective removal of heavy metal cations, e.g. Cu²⁺, Cd²⁺, Cr⁶⁺, Ni²⁺, Pb²⁺, and Zn²⁺ from aqueous solutions has been demonstrated²⁴, and the removal of low-level radionuclides, from liquid streams²⁵. In that latter application, a “feeder layer” of soil is suspended above the stream through which plants grow, from which the plant roots extend downward into the water. In this way, fertilizer can be used to help the plants to grow, while avoiding adding to the contamination of the stream, while the latter is cleansed of heavy metal cations²⁶. Rhizofiltration is cost-effective when large volumes of water must be treated containing low concentrations of contaminants. Inclusive of the costs of the capital outlay and final waste disposal, the cost of removing radionuclides from water using sunflowers was reckoned (at 1996 prices) at $2─6 per thousand gallons of water treated²⁷.

References.

(1) http://www.clu-in.org/download/citizens/a_citizens_guide_to_bioremediation.pdf

(2) http://www.ars.usda.gov/is/ar/archive/jun00/soil0600.htm

(3) http://en.wikipedia.org/wiki/Bioremediation

(4) Meagher, R.B. (2000). "Phytoremediation of toxic elemental and organic pollutants." Current Opinion in Plant Biology 3, 153.

(5) Diaz E (editor). (2008). Microbial Biodegradation: Genomics and Molecular Biology (1st ed.). Caister Academic Press.

(6) Brim, H. et al. (2000) "Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments." Nature Biotechnology 18, 85.

(7) Librando, V. and Pappalardo, M.J. (2013) “in silico bioremediation of polycyclic aromatic hydrocarbon: A frontier in environmental chemistry.” Mol. Graph. Model. May 14;44C:1-8. doi: 10.1016/j.jmgm.2013.04.011. [Epub ahead of print]

(8) Heider, J. and Rabus, R. (2008) "Genomic Insights in the Anaerobic Biodegradation of Organic Pollutants." Microbial Biodegradation: Genomics and Molecular Biology. Caister Academic Press. ISBN 978-1-904455-17-2.

(9) Cupples, A. M., Sanford, R.A. and Sims, G.K. (2005) “Dehalogenation of Bromoxynil (3,5-Dibromo-4-Hydroxybenzonitrile) and Ioxynil (3,5-Diiodino-4-Hydroxybenzonitrile) by Desulfitobacterium chlororespirans.” Appl. Env. Micro. 71, 3741.

(10) Villemur, R. (2013) “The pentachlorophenol-dehalogenating Desulfitobacterium hafniense strain PCP-1.” Phil. Trans. Roy. Soc. B. 368, 1616.

(11) Ding, C., Chow, W.L., Lee,L.K. and He,J.(2013)”Isolation of Acetobacterium sp. 

Strain AG, Which Reductively Debrominates Octa- and Pentabrominated Diphenyl Ether 

Technical Mixtures.” J. Appl. Environ. Microbiol. 79,1110.

(12) O'Loughlin, E. J, Traina, S.J. and Sims, G.K. (2000) “Effects of sorption on the 

biodegradation of 2-methylpyridine in aqueous suspensions of reference clay minerals.” 

Environ. Toxicol. and Chem. 19, 2168.

(13) Parales, R.E. et al. (2008). "Bioavailability, Chemotaxis, and Transport of Organic 

Pollutants." Microbial Biodegradation: Genomics and Molecular Biology. Caister Academic 

Press. ISBN 978-1-904455-17-2.

(14) http://www.biotecharticles.com/Applications-Article/Potential-Hydrocarbonoclastic-Bacteria-730.html

(15) Dracopoulos, D.C., and Piccoli, R. (2010) “Bioreactor control by genetic programming”, Parallel Problem Solving from Nature, PPSN XI Lecture Notes in Computer Science, 6239, 181.

(16) http://en.wikipedia.org/wiki/Mycoremediation

(17) http://www.fungi.com/blog/items/helping-the-ecosystem-through-mushroom-cultivation.html

(18) Burken, J.G. (2004), "2. Uptake and Metabolism of Organic Compounds: Green-Liver Model", in McCutcheon, S.C.; Schnoor, J.L. (Eds.), Phytoremediation: Transformation and Control of Contaminants, A Wiley-Interscience Series of Texts and Monographs, Hoboken, NJ: John Wiley, p. 59, doi:10.1002/047127304X.ch2, ISBN 0-471-39435-1

(19) http://en.wikipedia.org/wiki/Phytoremediation

(20) http://www.unep.or.jp/Ietc/Publications/Freshwater/FMS2/2.asp

(21) http://tede.ibict.br/tde_busca/arquivo.php?codArquivo=431

(22) Dushenkov, S. (2003) “Trends in phytoremediation or radionuclides.” Plant and Soil, 249, 167.

(23) http://en.wikipedia.org/wiki/Rhizofiltration

(24) EPA, (1998) “A Citizen's Guide to Phytoremediation,.” U.S. Environmental Protection Agency, Office of Solid Waste and Emergency Response,” EPA 542-F-98-011, August.

(25) Dushenkov, V., Motto, H., Raskin, I. and Nanda Kumar, P.B.A. (1995) "Rhizofiltration: the Use of Plants to Remove Heavy Metals From Aqueous Streams." Environmental Science Technology 30, 1239.

(26) Raskin, I., Smith, R.D. and Salt, D.E. (1997) "Phytoremediation of Metals: Using Plants to Remove Pollutants from the Environment." Current Opinion in Biotechnology. 8, 221.

(27) Cooney, C. M. (1996) "Sunflowers Remove Radionuclides From Water in Ongoing Phytoremediation Field Tests." Environmental Science and Technology 30, 194.

Radiation, Free Radicals and Disease.

This text is based on a lecture, "Radiation, Free Radicals and Disease," first delivered by Professor Chris Rhodes at Kingston University, London on Wednesday, November the 27th, 2013, and which he gave subsequently at the Slovak Technical University in Bratislava (24th June, 2014), and the University of Constantine the Philosopher in Nitra, Slovakia in 2014 (25th June, 2014).

Radiation breaks down water in living cells into dangerously reactive free radicals which subject our bodies to a continual assault, attacking essential molecules of life such as DNA and proteins. Since radiation has been around for as long as the Earth has, we have fortunately evolved efficient defense systems called antioxidants, which generally intercept the radicals before they do too much damage. The energy spectrum of radiation spans the range from radio-waves, at fairly low energies, through the microwave, infra red, visible and ultra-violet wavelengths of light, and into the X-ray, gamma-ray and cosmic-ray regions.

The latter constitute "ionising radiations" which are of relatively high energies, up to millions of electron volts. The disintegration of radioactive nuclei too, results in energetic particles which can also ionise molecules, such as beta-rays (high energy electrons) and alpha-particles, along with gamma-rays. Indeed most nuclear disintegrations release gamma rays as the decay ("daughter") product nucleus is formed in an excited state and emits a gamma ray on returning to its state of lowest energy.

The word "radiation" refers to the process of emitting energy in the form of rays or particles, which are themselves often called "radiations". In 1895, Wilhelm Roentgen was the first to identify (though probably not discover as vacuum "Crookes" tubes had been around for several decades) X-rays, streaming from the "positive electrode" in such cathode ray tubes. Cathode rays are energetic electrons which "boil-off" from the surface of an electrically heated cathode, and on striking a (positive) electrode, to which they are attracted by virtue of their own negative charge, excite atoms in the target, which emit energetic radiations known as X-rays.

Within months of Roentgen's report, Antoine Henri Becquerel had identified natural radioactivity from uranium ore, making the discovery that the mineral could darken a photographic plate, even when the latter was wrapped in paper, and so the curious radiations had a peculiar power of penetration. In 1896, the first x-ray "photograph" was taken, in fact of Roentgen's wife's left hand - the bones are clearly visible, as indeed is her wedding ring, since the rays are stopped by denser material, thus permitting structural differentiation in living tissue.

I wonder why Roentgen didn't X-ray his own hand, but left this privilege to his wife! Perhaps he wasn't entirely sure that x-rays were harmless and used her as a guinea-pig; but more likely, he did consider it a privilege, as radiations were to be revered for the next few decades as being possessed by almost miraculous properties. Indeed, some tens of thousands of "radium emanators" were sold and installed the houses mostly of Americans, so they could "invigorate" drinking water and thus receive the "beneficial" consequences of "radium emanation", i.e. what we now call Radon and try to protect ourselves from at all costs. Remarkably, radium suppositories were also sold, with the intention of re-invigorating the flagging manhood.

X-rays were put to generalised use in 1897 by the U.S. Army in the Spanish- American War to locate bullets and shrapnel in wounded soldiers.

In 1898, Marie Curie discovered that thorium too emitted "uranium radiation", and coined the term "radioactivity" to describe the phenomenon. In this same year, Marie and Pierre Curie manged to identify both the elements polonium (named after her native Poland) and radium. Polonium is a decay product of radium and both are members of the decay-chain leading down from uranium-238. The elements were "discovered" using spectroscopy, since both exhibited spectral lines unknown for any other elements.

My casual annotation here trivialises the immensity of their accomplishment, which involved boiling, in 20 kilogram batches, 4 tonnes of uranium ore (pitchblende) in concentrated acids, and then separating out the constituent elements using probably the most laborious technique in classical chemistry: "fractional crystallisation". All the work was done in an unheated shed, which, as they came closer to their goal, was lit at night by the green glow from the radioactive salts in petri-dishes spread out over the simple wooden tables that were its furniture. Marie Curie described herself as "often physically exhausted" at the end of a day's work, no doubt as she, a slightly built woman, handled half-hundredweight quantities of materials, and stirred them with a heavy iron bar in cauldrons of boiling acid.

It is likely too, that her strength was sapped by exposure to ionising radiation from these elements from which there was no protection at all, and which eventually took her life in the form of aplastic pernicious anemia (a form of leukemia). Yet, she described those four years spent working "in the shed" with her husband as being "their happiest". I imagine an almost spiritual euphoria of idealism, purpose and love, which only a precious few of us experience.

 Marie Curie was awarded the Nobel Prize for Physics, along with Pierre Curie and Becquerel, in 1903. In 1902, she isolated pure radium by electrolysing a solution of radium chloride (RaCl2) in water using a mercury cathode: the mercury was distilled-off, leaving 100 milligrams of metallic radium. This led to her being awarded a second Nobel Prize in 1910.

In 1899, Ernest Rutherford discovered "alpha-rays" and "beta-rays", as he termed them, differentiating between the two kinds by their differing penetrating power. Specifically, beta-rays (electrons) are stopped by a sheet of paper, but alpha-rays (helium nuclei and hence particles) require a sheet of aluminium to stop them. The lawyers were in quickly enough too, and also in 1899 the first malpractice suit award was made for x-ray "burns".

Though the public "miracle" of radiation had inaugurated a huge "healthy radon" industry, those working with radioactive materials and x-ray equipment had come to realise that there were serious adverse health-effects attendant to radiation. As early as 1902, it was shown that a dose of x-rays could cause death in a mammalian foetus. Both the Curies and Becquerel received radiation "burns" from handling radium. The connection was made between exposure to radiation and the development of tumours.

One particularly unfortunate pioneer was Clarence Dally, a fine experimentalist who was employed by the U.S. inventor Thomas Edison in making and experimenting with x-ray tubes. Dally suffered a steady contracting of tumours to both hands, which necessitated the amputation of his fingers, then hands and finally both arms. He finally died of generalised cancer, aged just 39. Radiation-induced tumours are a result of free radicals causing damage to DNA in cells, which mostly (80%) arise from the breakdown ("radiolysis") of water in the sheath surrounding the DNA double helix, while the remaining 20% is from direct damage to the DNA molecules by ionising radiation.

The symptoms of "radiation sickness" are well known (vomiting, hair-loss and in high doses, an ultimate liquefaction of internal organs as proteolytic enzymes are spilled from breached cell interfaces, then death). The lethal dose to a human is around 500 - 1000 Rems (5 - 10 Sieverts). Many animals have been irradiated in "radiation biology" experiments, and in the 1950's a connection was made between the effects of exposure to x-rays and "oxygen poisoning", which deep-sea divers may experience under inopportune circumstances (accelerated metabolism, coma and even death). It was proposed that oxygen free radicals may be involved in both cases.

This led to the notion by Denham Harman in 1956 that the reason we age is that cells in the body are constantly attacked by free radicals formed from oxygen (which we breathe) and transition metals such as iron, and ultimately we die from accumulated wear and tear. In a similar manner to an old car, which rusts from exposure of oxygen radicals, so do we and eventually become "old bangers" and are finally shipped-off to the junk-yard!

As an extension of this "free radical theory of aging" is the notion that many diseases, including cancer, arthritis and cardiovascular disease, are the result of injury from oxygen free radicals. A huge industry has grown-up based around the concept of "antioxidants", and that taking supplements of antioxidants in the form of pills to some extent reduces the damage, and hence the likelihood of developing these illnesses. However, there is no conclusive evidence that antioxidant supplements do any good at all, whereas there is well documented data that taking too many or the wrong kinds can be positively harmful. In one trial of beta-carotene supplements in smokers in Finland, the intended three-year long trail was discontinued after just nine months because subjects began to show an elevation in levels of lung cancer by about 20%!

Since beta-carotene is thought to protect against cancer, this is most alarming, but it just shows that Nature is more complex than we comprehend, and the only hard medical statistical evidence is that eating 5 - 8 portions of fruit and vegetables per day does seem to correspond with a reduced level of cancer, heart disease and arthritis. This is usually referred to as the "Mediterranean Diet", but I wonder to what extent the benefit actually is provided by the "Mediterranean Lifestyle", that people who are warmer, less stressed and happier are less prone to these diseases than are those of us living in the colder, more societally frenetic northern countries. It is likely that the human body has been adapted by evolution to adjust the balance between ROS and antioxidants so finely that the intake of additional antioxidants has but a minor influence, and so the degree of oxidative damage is little reduced. In a way, it is reminiscent of the concept of ‘inbuilt obsolescence,’ that we cannot live forever and are designed not to.

To whit, the degree of protein oxidation (according to the infra-red C=O bond absorption intensity) in samples taken from species of vastly differential longevity (rats, rabbits, flies and humans) has been plotted as a function of lifespan fraction, and remarkably in all cases, all is fairly constant (well protected) until around the half-way point - i.e. around the age of 40 or so in the case of humans - whereupon a relentless upsurge occurs, as we progress toward its conclusion. In all probability, we are protected by Nature until we have passed our reproductive years, and then the segment of the code reads-through to conclude this brief ride on Spaceship Earth, making way for the next  and fresher generation.