Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

A research platform for artificial neural networks with applications in pediatric epilepsy

This dissertation established a state-of-the-art programming tool for designing and training artificial neural networks (ANNs) and showed its applicability to brain research. The developed tool, called NeuralStudio, allows users without programming skills to conduct studies based on ANNs in a powerful and very user friendly interface. A series of unique features has been implemented in NeuralStudio, such as ROC analysis, cross-validation, network averaging, topology optimization, and optimization of the activation function’s slopes. It also included a Support Vector Machines module for comparison purposes. Once the tool was fully developed, it was applied to two studies in brain research. In the first study, the goal was to create and train an ANN to detect epileptic seizures from subdural EEG. This analysis involved extracting features from the spectral power in the gamma frequencies. In the second application, a unique method was devised to link EEG recordings to epileptic and nonepileptic subjects. The contribution of this method consisted of developing a descriptor matrix that can be used to represent any EEG file regarding its duration and the number of electrodes. The first study showed that the inter-electrode mean of the spectral power in the gamma frequencies and its duration above a specific threshold performs better than the other frequencies in seizure detection, exhibiting an accuracy of 95.90%, a sensitivity of 92.59%, and a specificity of 96.84%. The second study yielded that Hjorth’s parameter activity is sufficient to accurately relate EEG to epileptic and non-epileptic subjects. After testing, accuracy, sensitivity and specificity of the classifier were all above 0.9667. Statistical tests measured the superiority of activity at over 99.99 % certainty. It was demonstrated that (1) the spectral power in the gamma frequencies is highly effective in locating seizures from EEG and (2) activity can be used to link EEG recordings to epileptic and non-epileptic subjects. These two studies required high computational load and could be addressed thanks to NeuralStudio. From a medical perspective, both methods proved the merits of NeuralStudio in brain research applications. For its outstanding features, NeuralStudio has been recently awarded a patent (US patent No. 7502763).

A Research Platform for Artificial Neural Networks with Applications in Pediatric Epilepsy

This dissertation established a state-of-the-art programming tool for designing and training artificial neural networks (ANNs) and showed its applicability to brain research. The developed tool, called NeuralStudio, allows users without programming skills to conduct studies based on ANNs in a powerful and very user friendly interface. A series of unique features has been implemented in NeuralStudio, such as ROC analysis, cross-validation, network averaging, topology optimization, and optimization of the activation function’s slopes. It also included a Support Vector Machines module for comparison purposes. Once the tool was fully developed, it was applied to two studies in brain research. In the first study, the goal was to create and train an ANN to detect epileptic seizures from subdural EEG. This analysis involved extracting features from the spectral power in the gamma frequencies. In the second application, a unique method was devised to link EEG recordings to epileptic and non-epileptic subjects. The contribution of this method consisted of developing a descriptor matrix that can be used to represent any EEG file regarding its duration and the number of electrodes. The first study showed that the inter-electrode mean of the spectral power in the gamma frequencies and its duration above a specific threshold performs better than the other frequencies in seizure detection, exhibiting an accuracy of 95.90%, a sensitivity of 92.59%, and a specificity of 96.84%. The second study yielded that Hjorth’s parameter activity is sufficient to accurately relate EEG to epileptic and non-epileptic subjects. After testing, accuracy, sensitivity and specificity of the classifier were all above 0.9667. Statistical tests measured the superiority of activity at over 99.99 % certainty. It was demonstrated that 1) the spectral power in the gamma frequencies is highly effective in locating seizures from EEG and 2) activity can be used to link EEG recordings to epileptic and non-epileptic subjects. These two studies required high computational load and could be addressed thanks to NeuralStudio. From a medical perspective, both methods proved the merits of NeuralStudio in brain research applications. For its outstanding features, NeuralStudio has been recently awarded a patent (US patent No. 7502763).

Geopiracy: Patent law, climate change, and geoengineering

Patent law is a regime of intellectual property, which provides exclusive rights regarding scientific inventions, which are novel, inventive, and useful. There has been much debate over the limits of patentable subject matter relating to emerging technologies. The Supreme Court of the US has sought to rein in the expansive interpretation of patentability by lower courts in a series of cases dealing with medical information (Prometheus), finance (Bilski), and gene patents (Myriad). This has led to a reinvigoration of the debate over the boundaries of patentable subject matter. There has been controversy about the rise in patenting of geoengineering - particularly by firms such as Intellectual Ventures.

Access to medicine and the dangers of patent linkage : lessons from Bayer Corp v. Union of India

In February 2010, the Delhi High Court delivered its decision in Bayer Corp v Union of India in which Bayer had appealed against an August 2009 decision of the same court. Both decisions prevented Bayer from introducing the concept of patent linkage into India’s drug regulatory regime. Bayer appealed to the Indian Supreme Court, the highest court in India, which agreed on 2 March 2010 to hear the appeal. Given that India is regarded as a global pharmaceutical manufacturer of generic medications, how its judiciary and government perceive their international obligations has a significant impact on the global access to medicines regime. In rejecting the application of patent linkage, the case provides an opportunity for India to further acknowledge its international human rights obligations.

Patent-busting: The public patent foundation, gene patents and the Seed Wars

This chapter considers the Public Patent Foundation as a novel institution in the patent framework. It contends that such a model can play a productive role in challenging the validity of high-profile patents; working as an amicus curiae in significant court cases; and also promoting patent law reform. However, there are limits to the ‘patent-busting’ of the Foundation. The not-for-profit legal services organization has only had the time and resources to challenge a number of noteworthy patents. Other jurisdictions – such as Australia – lack such public-spirited "patent-busting" entities. This chapter considers a number of key disputes involving the Public Patent Foundation. Part I examines the role of the Public Patent Foundation in the landmark dispute over Myriad Genetics’ patents in respect of breast cancer and ovarian cancer. Part II considers the role of the Public Patent Foundation in litigation between organic farmers and Monsanto. Part III examines the role of the Public Patent Foundation in larger debates about patent law reform in the United States – particularly looking at the Leahy-Smith America Invents Act 2011 (US). The conclusion contends that the patent-busting model of the Public Patent Foundation should be emulated in respect of other technological fields, and other jurisdictions – such as Australia. The initiative could also be productively applied to other forms of intellectual property – such as trade mark law, designs law, plant breeders’ rights, plant breeders’ rights, and access to genetic resources.

After the gold rush: Patent law and biological inventions

In response to scientific breakthroughs in biotechnology, the development of new technologies, and the demands of a hungry capitalist marketplace, patent law has expanded to accommodate a range of biological inventions. There has been much academic and public debate as to whether gene patents have a positive impact upon research and development, health-care, and the protection of the environment. In a satire of prevailing patenting practices, the English poet and part-time casino waitress, Donna MacLean, sought a patent application - GB0000180.0 - in respect of herself. She explained that she had satisfied the usual patent criteria - in that she was novel, inventive, and useful: It has taken 30 years of hard labor for me to discover and invent myself, and now I wish to protect my invention from unauthorized exploitation, genetic or otherwise. I am new: I have led a private existence and I have not made the invention of myself public. I am not obvious (2000: 18). MacLean said she had many industrial applications. ’For example, my genes can be used in medical research to extremely profitable ends - I therefore wish to have sole control of my own genetic material' (2000: 18). She observed in an interview: ’There's a kind of unpleasant, grasping, greedy atmosphere at the moment around the mapping of the human genome ... I wanted to see if a human being could protect their own genes in law' (Meek, 2000). This special issue of Law in Context charts a new era in the long-standing debate over biological inventions. In the wake of the expansion of patentable subject matter, there has been great strain placed upon patent criteria - such as ’novelty', ’inventive step', and ’utility'. Furthermore, there has been a new focus upon legal doctrines which facilitate access to patented inventions - like the defence of experimental use, the ’Bolar' exception, patent pooling, and compulsory licensing. There has been a concerted effort to renew patent law with an infusion of ethical principles dealing with informed consent and benefit sharing. There has also been a backlash against the commercialisation of biological inventions, and a call by some activists for the abolition of patents on genetic inventions. This collection considers a wide range of biological inventions - ranging from micro-organisms, plants and flowers and transgenic animals to genes, express sequence tags, and research tools, as well as genetic diagnostic tests and pharmaceutical drugs. It is thus an important corrective to much policy work, which has been limited in its purview to merely gene patents and biomedical research. This collection compares and contrasts the various approaches of a number of jurisdictions to the legal problems in respect of biological inventions. In particular, it looks at the complexities of the 1998 European Union Directive on the Legal Protection of Biotechnological Inventions, as well as decisions of member states, such as the Netherlands, and peripheral states, like Iceland. The edition considers US jurisprudence on patent law and policy, as well as recent developments in Canada. It also focuses upon recent developments in Australia - especially in the wake of parallel policy inquiries into gene patents and access to genetic resources.

Patent law and biological inventions

In response to scientific breakthroughs in biotechnology, the development of new technologies, and the demands of a hungry capitalist marketplace, patent law has expanded to accommodate a range of biological inventions. There has been much academic and public debate as to whether gene patents have a positive impact upon research and development, health-care, and the protection of the environment. In a satire of prevailing patenting practices, the English poet and part-time casino waitress, Donna MacLean, sought a patent application - GB0000180.0 - in respect of herself. She explained that she had satisfied the usual patent criteria - in that she was novel, inventive, and useful: It has taken 30 years of hard labor for me to discover and invent myself, and now I wish to protect my invention from unauthorized exploitation, genetic or otherwise. I am new: I have led a private existence and I have not made the invention of myself public. I am not obvious (2000: 18). MacLean said she had many industrial applications. 'For example, my genes can be used in medical research to extremely profitable ends - I therefore wish to have sole control of my own genetic material' (2000: 18). She observed in an interview: 'There's a kind of unpleasant, grasping, greedy atmosphere at the moment around the mapping of the human genome ... I wanted to see if a human being could protect their own genes in law' (Meek, 2000). This special issue of Law in Context charts a new era in the long-standing debate over biological inventions. In the wake of the expansion of patentable subject matter, there has been great strain placed upon patent criteria - such as 'novelty', 'inventive step', and 'utility'. Furthermore, there has been a new focus upon legal doctrines which facilitate access to patented inventions - like the defence of experimental use, the 'Bolar' exception, patent pooling, and compulsory licensing. There has been a concerted effort to renew patent law with an infusion of ethical principles dealing with informed consent and benefit sharing. There has also been a backlash against the commercialisation of biological inventions, and a call by some activists for the abolition of patents on genetic inventions. This collection considers a wide range of biological inventions - ranging from micro-organisms, plants and flowers and transgenic animals to genes, express sequence tags, and research tools, as well as genetic diagnostic tests and pharmaceutical drugs. It is thus an important corrective to much policy work, which has been limited in its purview to merely gene patents and biomedical research. This collection compares and contrasts the various approaches of a number of jurisdictions to the legal problems in respect of biological inventions. In particular, it looks at the complexities of the 1998 European Union Directive on the Legal Protection of Biotechnological Inventions, as well as decisions of member states, such as the Netherlands, and peripheral states, like Iceland. The edition considers US jurisprudence on patent law and policy, as well as recent developments in Canada. It also focuses upon recent developments in Australia - especially in the wake of parallel policy inquiries into gene patents and access to genetic resources.

Vanquishing the'Patent Trolls'

Patent law provides exclusive rights to exploit scientific inventions, which are novel, inventive, and useful. The regime is intended to promote innovation, investment in research and development, and access to scientific information. In recent times, there have been concerns that the patent system has been abused by opportunistic companies known by the phrase “patent trolls”. It has been alleged that such entities have stunted innovation and spurred unnecessary patent litigation. Adam Jaffe and Josh Lerner discuss such pathologies of patent law in their book, Innovation and Its Discontents: How our Broken Patent System is Endangering Innovation and Progress, and What To Do About It. James Bessen and Michael Meurer have addressed such concerns in their recent text, Patent Failure: How Judges, Bureaucrats, and Lawyers Put Innovators at Risk. There have been particular fears about the rise of “patent trolls” in the field of information technology. Peter Dekin, an assistant general counsel at Intel, used the phrase “patent troll” to describe firms, which acquired patents only to extract settlements from companies on dubious infringement claims.

Turing and the Trans-Pacific Partnership: Intellectual Property, Public Health, and Access to Essential Medicines

A recent controversy in the United States over drug pricing by Turing Pharmaceuticals AG has raised larger issues in respect of intellectual property, access to medicines, and the Trans-Pacific Partnership (TPP). In August 2015, Turing Pharmaceuticals AG – a private biopharmaceutical company with offices in New York, the United States, and Zug, Switzerland - acquired the exclusive marketing rights to Daraprim in the United States from Impax Laboratories Incorporated. Martin Shkreli, Turing’s Founder and Chief Executive Officer, maintained: “The acquisition of Daraprim and our toxoplasmosis research program are significant steps along Turing’s path of bringing novel medications to patients with serious disorders, some of whom often go undiagnosed and untreated.” He emphasised: “We intend to invest in the development of new drug candidates that we hope will yield an even better clinical profile, and also plan to launch an educational effort to help raise awareness and improve diagnosis for patients with toxoplasmosis.” In September 2015, there was much public controversy over the decision of Martin Shkreli to raise the price of a 62 year old drug, Daraprim, from $US13.50 to $US750 a pill. The drug is particularly useful in respect to the treatment and prevention of malaria, and in the treatment of infections in individuals with HIV/AIDS. Daraprim is listed on the World Health Organization’s (WHO) List of Essential Medicines. In the face of much criticism, Martin Shkreli has said that he will reduce the price of Daraprim. He observed: “We've agreed to lower the price on Daraprim to a point that is more affordable and is able to allow the company to make a profit, but a very small profit.” He maintained: “We think these changes will be welcomed.” However, he has been vague and ambiguous about the nature of the commitment. Notably, the lobby group, Pharmaceutical Research and Manufacturers of America (PhARMA), disassociated itself from the claims of Turing Pharmaceuticals. The group said: “PhRMA members have a long history of drug discovery and innovation that has led to increased longevity and improved lives for millions of patients.” The group noted: “Turing Pharmaceutical is not a member of PhRMA and we do not embrace either their recent actions or the conduct of their CEO.” The biotechnology peak body Biotechnology Industry Organization also sought to distance itself from Turing Pharmaceuticals. A hot topic: United States political debate about access to affordable medicines This controversy over Daraprim is unusual – given the age of drug concerned. Daraprim is not subject to patent protection. Nonetheless, there remains a monopoly in respect of the marketplace. Drug pricing is not an isolated problem. There have been many concerns about drug pricing – particularly in respect of essential medicines for HIV/AIDS, tuberculosis, and malaria. This recent controversy is part of a larger debate about access to affordable medicines. The dispute raises larger issues about healthcare, consumer rights, competition policy, and trade. The Daraprim controversy has provided impetus for law reform in the US. US Presidential Candidate Hillary Clinton commented: “Price gouging like this in this specialty drug market is outrageous.” In response to her comments, the Nasdaq Biotechnology Index fell sharply. Hillary Clinton has announced a prescription drug reform plan to protect consumers and promote innovation – while putting an end to profiteering. On her campaign site, she has emphasised that “affordable healthcare is a basic human right.” Her rival progressive candidate, Bernie Sanders, was also concerned about the price hike. He wrote a letter to Martin Shkreli, complaining about the price increase for the drug Daraprim. Sanders said: “The enormous, overnight price increase for Daraprim is just the latest in a long list of skyrocketing price increases for certain critical medications.” He has pushed for reforms to intellectual property to make medicines affordable. The TPP and intellectual property The Daraprim controversy and political debate raises further issues about the design of the TPP. The dispute highlights the dangers of extending the rights of pharmaceutical drug companies under intellectual property, investor-state dispute settlement, and drug administration. Recently, the civil society group Knowledge Ecology International published a leaked draft of the Intellectual Property Chapter of the TPP. Knowledge Ecology International Director, James Love, was concerned the text revealed that the US “continues to be the most aggressive supporter of expanded intellectual property rights for drug companies.” He was concerned that “the proposals contained in the TPP will harm consumers and in some cases block innovation.” James Love feared: “In countless ways, the Obama Administration has sought to expand and extend drug monopolies and raise drug prices.” He maintained: “The astonishing collection of proposals pandering to big drug companies make more difficult the task of ensuring access to drugs for the treatment of cancer and other diseases and conditions.” Love called for a different approach to intellectual property and trade: “Rather than focusing on more intellectual property rights for drug companies, and a death-inducing spiral of higher prices and access barriers, the trade agreement could seek new norms to expand the funding of medical research and development (R&D) as a public good, an area where the US has an admirable track record, such as the public funding of research at the National Institutes of Health (NIH) and other federal agencies.” In addition, there has been much concern about the Investment Chapter of the TPP. The investor-state dispute settlement regime would enable foreign investors to challenge government policy making, which affected their investments. In the context of healthcare, there is a worry that pharmaceutical drug companies will deploy their investor rights to challenge public health measures – such as, for instance, initiatives to curb drug pricing and profiteering. Such concerns are not merely theoretical. Eli Lilly has brought an investor action against the Canadian Government over the rejection of its drug patents under the investor-state dispute settlement regime of the North American Free Trade Agreement (NAFTA). The Health Annex to the TPP also raises worries that pharmaceutical drug companies will able to object to regulatory procedures in respect of healthcare. It is disappointing that the TPP – in the leaks that we have seen – has only limited recognition of the importance of access to essential medicines. There is a need to ensure that there are proper safeguards to provide access to essential medicines – particularly in respect of HIV/AIDs, malaria, and tuberculosis. Moreover, there must be protection against drug profiteering and price gouging in any trade agreement. There should be strong measures against the abuse of intellectual property rights. The dispute over Turing Pharmaceuticals AG and Daraprim is an important cautionary warning in respect of some of the dangers present in the secret negotiations in respect of the TPP. There is a need to preserve consumer rights, competition policy, and public health in trade negotiations over an agreement covering the Pacific Rim.