Marine Threat: Pollution
Marine Threat: Pollution
Consultants: Elif Demir-Hilton & Kevin Webb
Pollution is a major threat to the health of our oceans, and one of the greatest challenges for mitigation efforts due to the scale and diversity of sources that are tightly coupled to human activity and the global economy. It is essential that we develop ways to eliminate ocean pollution at the source, through new technologies that replace polluting industrial practices and products. In addition, to prevent a major loss of biodiversity, we must make methods of cleaning up the existing contamination of marine environments a priority. Two major types of pollution discussed in this chapter, industrial waste streams, as exemplified by crude oil spills, and solid plastic waste, could benefit from the application of biotechnology and genomics.
Marine pollution comes from a variety of sources and can have widespread and long-lasting effects on the ecosystem. Most marine pollution starts outside the ocean environment, as agricultural, industrial, urban runoff, wastewater, or even air pollution. Most human waste streams find their way to the ocean eventually. Once there, pollutants have a variety of effects, including acidification, eutrophication, and the accumulation of toxins in oceanic food webs (that then can make their way back to the terrestrial food webs). A comprehensive plan to cleanse the ocean environment must include efforts to reduce contamination of the land, fresh water, and air.
Petroleum pollution in marine environments is a particularly serious environmental concern, caused almost exclusively by human activity. Oil spills are among the most well publicized and dramatic pollution events. However, most of the oil pollution in the ocean is due to continuous small leaks from oil tanker ballasts, leaky oil pipelines, and improperly disposed of engine oil. The components of crude oil, such as polycyclic aromatic hydrocarbons, are toxic to marine life and are difficult to clean up. The restoration of ecological balance requires the development of effective ways to remediate oil-polluted environments.
Modern wastewater treatment can have a profound impact on the condition of discharges of pollutants and water quality in coastal waters. Unfortunately, sophisticated wastewater treatment facilities are costly to build and maintain. Also, contaminants of emerging concern are increasingly recognized as having profound impacts on the ecology of marine organisms. These contaminants include endocrine disruptors and other pharmaceuticals for which there are few recognized removal methods.
Nonpoint pollution, derived from many different sources and transferred as runoff from snowmelt or rain, is a particularly challenging management concern. Nonpoint pollution can contain toxic industrial chemicals, agricultural products like pesticides and fertilizers, pharmaceuticals from human wastewater, petroleum, and solid debris like plastic and dust. Plastics are largely a diffuse “non-point” problem. Notably, fishing nets are a significant source of plastic pollution. Conventional and highly targeted point source wastewater control methods are not effective. Instead, managers rely on retrofitting infrastructure to capture and passively “treat” non-point sources of pollution.
The increased levels of carbon in the atmosphere due to the burning of fossil fuels is leading to the acidification of marine environments as carbon dioxide is dissolved into ocean waters. This effect is expected to continue along with overall climate change and will particularly affect marine wildlife. If the ocean becomes too acidic, the calcified shells of bivalves and corals with may begin to dissolve (Feely et al. 2009). If these key species are lost, dramatic degradation of ocean ecosystems will quickly follow. Biotechnologies that lead to a reduction in carbon dioxide production or that can capture carbon from the atmosphere are actively being sought to reverse the effect of air pollution on our oceans. Studies have documented a localized ameliorating effect on acidification from submerged aquatic vegetation.
The effectiveness of control and remediation of pollution discharges are wildly disparate around the world. This largely depends on the level of development and standard of living in a country. Even for the most developed countries, new biologically-based remediation efforts hold promise as particularly beneficial innovations to pursue. These advantages stem from the ability for biological methods to remediate pollutants for which remediation methods have proved challenging for more conventional treatment systems.
Intrinsic processes of pollution bio-remediation generally involve the metabolic breakdown of introduced waste chemicals by organisms native to the environment. While this natural process of bioremediation is effective, it is slow and toxins can persist in the food web for a long time. Recent biotechnological advances, particularly in the area of synthetic biology, now offer the potential to improve the efficiency of bioremediation and pollution sensing, both in the ocean and at the pollution sources. However, these technologies are only beginning to be explored in complex environments outside of the lab. Safety concerns and regulatory hurdles will need to be overcome before these new solutions can be fully deployed. These technologies are also being investigated as part of efforts to drastically reduce the amount of pollution produced by human activities, which is ultimately the only way that we will save the oceans if the human population and economy continue to expand.
The rate of bioremediation by microbes in a contaminated environment can be limited by the availability of nutrients and oxygen. Biostimulation is a method to encourage faster bioremediation by supplementing the contaminated site with the needed nutrients, similar to providing fertilizer to improve the growth of plants. The advantages of this method are that they make use of the native ecosystem, avoiding the introduction of foreign organisms. However, the ability to spread biostimulants over large areas, especially in marine environments where dilution can be a problem, is a major limitation. Furthermore, the added nutrients can also stimulate growth of competitors or harmful algae that either do not help the bioremediation process or harm native wildlife, resulting in a net neutral or even a net negative effect.
Bioaugmentation is a more aggressive approach that involves seeding a contaminated environment with microbes that can mediate the breakdown of the pollution. For this to be successful, the introduced organisms must be able to break down all the components of the pollution, which is a particular challenge in the case of crude oil spills. Furthermore, the introduced bioremediator must thrive in the potentially hostile contaminated environment alongside the native organisms. Therefore, this technique requires the use of microbial strains or microbial consortia that have been well-adapted to growth in the environment of interest. They are typically isolated from sites that have frequent contamination events, like near leaky oil pipes, or the sites of past oil spills.
Pathway Mining and Metabolic Engineering
As mentioned above, oil-degrading microbes are often first identified in contaminated environments, or by virtue of particular genes discovered through genome sequencing efforts. However, few strains can degrade all constituents of crude oil, or thrive in a range of natural environments that may require remediation, such as sand, soil, and fresh and marine water. This has led industry and academics to the idea of using genetic engineering to create new strains of oil-degraders, sometimes called “superbugs”, that possess metabolic pathways for multiple hydrocarbon substrates. In fact, the first patent ever filed for an engineered microbe was in 1971, for a strain of Pseudomonas engineered with four oil-degrading genes from other strains (Environment: oil-eating bug. Time Magazine 1975. Time.). This first example had a 10-100-fold faster oil remediation rate relative to naturally occurring soil bacteria.
Over the last four decades of molecular biology, a large catalog of metabolic parts has been generated from many organisms spanning many domains of life. The universality of genetics allows any one of these parts to be used by any organism that encodes it. Bioinformatic tools have been created to enable metabolic engineers to systematically explore the huge catalog of parts available to generate new metabolic pathways that will transform pollutant-X into safe product-Y. Because of the modularity of these pathways and the conservation of the enzymes for each intermediate reaction type, synthetic metabolism is just a matter of plug-and-play. Likewise, the pathway mining approach used to identify oil-degrading pathways is dependent on the number of sequenced genomes and the ability of bioinformatic algorithms to spot relevant patterns in the gene sequences or genomic structures. Similar efforts to uncover natural biodegradation pathways of other industrial pollutants based on genomic sequences and structure could enable the discovery of new bioremediation strains or communities from metagenomic data. The major challenges to these synthetic biology approaches are in scaling the manufacturing of these engineered microbes, and in gaining approval for environmental release.
To date, most engineered microbes have been designed for use in closed environments, such as large fermenters. The controlled environment protects the modified microbes from harsh environmental insults that might inhibit function and also prevents the release of these GMOs into the natural environment. In order to adapt engineered microbes to in situ bioremediation applications, several innovations will be required. First, the host strain will need to be able to live in the natural environment of interest, which will be polluted with one or more contaminants, but will also contain competitors and potentially predators. In the case of marine environments, dilution of the pollutants will also be a challenge. The engineered strain will need to perform optimally under these environmental constraints. Conversely, the safe release of engineered microbes into natural environments will be a concern, especially if they are designed to thrive in those conditions. Engineered microbes will need to include safety mechanisms that control their spread beyond what is intended for their bioremediation purposes. Several techniques now exist that could prepare engineered microbes for in situ remediation in the near future.
Designer Pollution-Eating Microbes
The new tools for metabolic engineering described above have renewed the commercial potential for bioremediation strategies involving engineered microbes. It is now possible to rapidly screen thousands of potential pathways, both natural and synthetic, for a given reactant and product. Today, there are many aspiring companies focused on upcycling industrial waste streams, some relying on designer microbes to convert trash to treasure. For example, a startup called Lanzatech is developing technology to use carbon-rich gas waste streams from steel manufacturing, agricultural processes, and oil refining, as a carbon source for the biosynthesis of valuable commodities, like chemical precursors, and biofuels. Their process relies on engineered microbes that can ferment waste molecules, which are otherwise destined to become a source of pollution, much the way microbes can convert sugar into ethanol in a brewery. Lanzatech estimates that by feeding a factory waste stream into its bioreactors, they can reduce pollution emissions by up to 85%, and produce a valuable product in the process.
Many types of pollution from agricultural, industrial, or military waste streams are made of xenobiotic compounds, which are compounds not found in nature. Although these molecules can be substrates for biodegradation, it is rare for the complete biodegradation pathway to be encoded in the genome of a single microbe. Metabolic engineering has been used to create strains of the soil microbe, Pseudomonas putida, that can simultaneously degrade multiple pesticides that are commonly used together, such as methyl parathion and c-Hexachlorocyclohexane, and carbofuran and chlorpyrifos (Gong et al. 2016).
Adaptive Laboratory Evolution (ALE)
One pragmatic approach to engineering strains that can thrive and function in sub-optimal environments is to take advantage of yet another universal feature of biology: the ability to adapt to new conditions through evolution. In nature, the adaptive evolution of a population of microbes can take decades; however, the small size and short generation times of microbes allow researchers to shorten evolutionary time dramatically in the lab by introducing increasing amounts of selective pressure with frequent artificial selection events. This approach has been successfully applied to enzymes numerous times in the creation of commercially-viable production strains for valuable molecules, like pharmaceuticals. The same approach is readily adapted at genome scale for bioremediation applications. Furthermore, researchers have begun to apply ALE to multi-strain consortia of microbes, which may be able to collectively degrade complex mixtures of contaminants, such as crude oil. The major challenge will be in the ability to simulate contaminated natural environments in a closed laboratory setting in order to select for genotypes that can thrive in those environmental conditions. This will be particularly challenging for marine environments.
The ability to reliably sense pollution in the environment at low levels is valuable for protecting ecosystems and for monitoring the bioremediation of contaminated sites. Living cells naturally have acute sensory systems for detecting chemicals in their immediate surroundings. These natural abilities can be harnessed to create highly sensitive and specific environmental sensors for industrial toxins and pollutants, including hydrocarbons, heavy metals, and organic compounds (Belkin 2016).
Engineered microbial bioreporters are inexpensive, easy to use, and can be more sensitive than electrochemical alternatives. These reporters are already used widely for medicine, biology, toxicology, drug screening, and water quality testing, and several field tests have demonstrated their ability to detect pollutants in natural environments. Multiple bioreporters can be used in parallel in environments that are contaminated with complex mixtures, like crude oil. For example, Tecon et al demonstrated the use of a suite of five bacterial bioreporters to monitor the levels of hydrocarbon mixtures in marine environments over periods of 7-10 days. The results from the bioreporters were comparable to those obtained by chemical analytic methods, both in the concentration and timing of analyte detection after the oil spill. The authors concluded that this suite of bioreporters could constitute a simple analytical tool to measure the time scale of oil spills.
Low Anticipated Return on Investment
As described above, the ability to engineer microbes with desired metabolic and life-style properties has been greatly increased by affordable genomic sequencing and genetic engineering tools. It is now possible to create a microbe that can both thrive in an environment of interest and possess the metabolic genes necessary to degrade complex mixtures of toxins, like crude oil. However, these engineered biological systems are not being commercialized for bioremediation applications as fast as they are for other industries, such as fermentative biosynthesis. This may be for economic reasons; there are simply more commercial opportunities in the bioproduction of commodity chemicals.
Additionally, steep regulatory hurdles exist for the environmental release of engineered organisms, which is a requirement for in situ bioremediation applications (Technology in Society 32 (2010) 331–335). The same public concern, policy, and governance that will regulate the release of GMOs for control of invasive species will also apply to bioremediation technology.
Several advances in the way that engineered oil-remediating strains are designed and tested prior to release could alleviate concerns and speed the time to practical use. First, redundant genetically encoded safety circuits should be a standard design feature of any strain that could be considered for release to ensure that growth can be limited to the time and place where the activity is needed. Several genetic systems for growth limitation currently exist, such as toxin/anti-toxin pairs, cell division counters, addiction pathways that make a microbe dependent on a nutrient additive (Wright et al. 2013), and environmental sensors that restrict growth to a particular location or context (Simon and Ellington 2016). Second, genetically engineered strains can be designed with one or more non-standard codons to prevent them from either picking up or donating genetic material to the natural environment (Rovner et al. 2015). Finally, experimental platforms that allow the safety and efficacy of engineered strains to be rigorously tested in relevant conditions need to be developed. This is particularly challenging for marine environments because of the complexity of the ecosystems, but perhaps even more necessary because of that.
Safety and Control
The solution to the safety concerns associated with genetically engineered organisms may actually be even more engineering to produce strains that contain internal safety mechanisms that prevent uncontrolled growth and genetic transfer with organisms in the surrounding environment. Precision genomic tools have progressed to the point that worrisome features of the early engineered strains, such as antibiotic resistance markers, are no longer necessary. Furthermore, techniques like multiplex-assisted genetic engineering (MAGE) make it now feasible to design microbes that use unique genetic codes and are therefore genetically isolated from the native organisms in the environment (Lajoie 2013). Finally, the field of synthetic biology has demonstrated the ability to design and build programmable gene circuits that behave more like natural genetic pathways with regulatory inputs and logic-based functions. These programmable cells can be designed to behave according to environmental signals and will have a lower risk of behaving inappropriately in a natural setting. It is expected that the potential for highly engineered microbes will open the door to fieldable bioremediation agents capable of rapidly degrading crude oil contamination in a range of environments. In addition, regulatory requirements may need to be updated to allow for field tests of engineered microbes that have built-in safety features.
SOLID WASTE: PLASTICS
Coastal communities are inundated with plastic waste and discarded fishing nets are a major source of plastic pollution globally. Currently there are five different known ‘floating islands’ of plastic circulating the Earth’s oceans, reaching up to several square kilometers in size. Plastic is extremely slow to degrade, and different types of plastics have different degradation rates and degradation pathways. Unfortunately, plastic degradation tends to just make it ever smaller into microplastics that then enter aquatic food chains where, like many pollutants, it tends to bioaccumulate up the food chain. Plastic affects marine life through direct consumption, as evidenced by beached whales and other wildlife frequently found with high volumes of plastic in their digestive tracts (Brate et al., 2016; Schuyler et al., 2016; Avery-Gomm et al., 2018). It is also having ecological cascading effects through impacts on food webs and the introduction of ecotoxicants that can affect endocrine and reproductive systems (Avio et al., 2017).
A promising approach for the treatment of marine pollution is microbial remediation. Microorganisms, particularly bacteria, can be bioengineered to optimally sense or break down and metabolize contaminants in the environment. Genomics, transcriptomics, proteomics and metabolomics can be applied in a systems biology approach to advance microbial remediation technology through synthetic biology.
Several technological innovations are being tested for efficacy in removing plastic waste from marine environments. Source reduction is an important strategy and several initiatives that target the reduction of the use of single use plastics are making strides. Other initiatives and start-up companies are seeking to reduce or replace the use of plastics in packaging.
In 2018, the launching of The Ocean Cleanup System 01 marked the first large-scale attempt to use a floating catchment system to localize and physically remove plastic waste from the environment. Despite some immediate setbacks, the company expects to move forward with testing system on the Great Pacific Garbage Patch. Smaller scale systems, such as the Seabin, have also been developed to filter plastic waste from calm marine environments such as ports and marinas. While these technologies hold potential to vastly reduce the amount of plastic waste accumulating in the ocean, neither offers a viable solution for permanent resolution of the plastic waste problem. However, if properly coupled around a bioremediation platform, technology and synthetic biology may be a viable and lasting solution to protect marine environments from plastic waste.
The most promising microbes for effective biodegradation of plastic are the Pseudomonas spp. (Table 1) although recent research has identified Ideonella sakaiensis 201-F6 as an efficient biodegrader of PET plastics (Yoshida et al., 2016). Additionally, a recent screen of microbial communities sampled in the Arctic and screened for plastic degradative abilities showed promise for Rhodococcus sp. of bacteria and several fungal species (Urbanek et al., 2017). However, rates of biodegradation are slow and not currently efficient to solve the ocean plastic problem. Various bottlenecks in the metabolic pathways, and influence of the environment and microbial metabolism have precluded the development of robust solutions. A systems biology and metabolic engineering approach is a key innovation strategy that needs to be explored.
Table borrowed from Whilkes and Aristilde (2017)
Synthetic biology coupled with metagenomic, comparative ‘omic’ and metabolic engineering approaches should be explored and developed to address the surmounting issue of plastic waste in oceans. It is important to note that, while this approach holds promise, the full realization of a bioremediation-based strategy to remove plastic waste from oceans remains distant. A major advantage of a synthetic biology approach to solid waste is that the pollution can be physically removed from the natural environment prior to treatment with engineered microbes. This eliminates the need for releasing engineered microbes into the natural environment, and reduces the safety and regulatory concerns that go along with that. Progress toward this goal will require 3 steps according to Dvorak et al. (2017) as highlighted below and represented in Fig 1:
- Model the appropriate degradation pathway for primary plastics in oceans using predictive software. This will require optimization for extracellular degradation of polymers into smaller chemicals, followed by transport into the cell and finally optimization of intracellular metabolism of plastic products.
- Identify the best host. Ocean plastics should be sampled to characterize microbial communities on biofilms. Newly developed sequencing techniques that take advantage of the three-dimensional structure of DNA (Hi-C Phase Genomics) allows researchers to generate, assemble and identify novel whole genomes from a metagenomic sample (Heger. 2014). That is, not only can they identify which bacteria are in a sample, but the entire genomic composition of each species present, better enabling understanding of how metabolic pathways are constructed within a single host, such that these can be targeted and engineered in highly specific ways, and combined into microbial consortia capable of overriding metabolic bottlenecks particular to any single microbial organism.
Apply computational and experimental tools to optimize metabolic pathways in the context of the host(s). Informed by the combination of various ‘omic’ analysis to identify the most suitable microbial host, whole genome and kinetic models can be coupled with genetic engineering techniques to tailor gene expression, reduce metabolic burden and optimize metabolic pathways (Whilkes and Aristilde 2017).
Figure borrowed from Dvorak et al. (2017)
Developing adequate bioremediation for plastic will be costly and requires highly specialized facilities. Further, advanced computational tools will need to be developed to integrate whole genome, transcriptome, and proteome data into metabolic pathway models. Once plastic is collected from the ocean, it may require manual processing or infrared sensors to sort the plastic into subtypes for handling by different microbes or microbial consortia. Collected plastic may also require pre-treatment to optimize microbial attachment. Substantial time and resources will be required to fully realize bioremediation and synthetic biology as a strategy to combat the ocean plastic problem. However, with well-directed funds, such a strategy could immensely improve conservation outcomes in marine environments.
The Ocean Cleanup—launched the Ocean Cleanup System 001 in September 2018. Despite initial functional failures, the system could provide a means to collect the waste which can then be collected into mobile bioremediation facilities onboard. This would alleviate environmental concerns over releasing bioengineered microbes into the natural environment, allowing containment and control of the engineering process.
Enoveo—ENOVEO was founded in 2008 and specializes in environmental microbiology, chemistry and biotechnologies. The result of collaboration between researchers at the University of Lyon and contaminated site management professionals, this company provides a range of services to facilitate the research and development of specific bioremediation challenges. They provide feasibility tests for custom projects, which to date include Petroleum hydrocarbons, PAHs, chlorinated solvents, polar solvents, PCBs, heterocyclic compounds, pesticides, chromium VI, BTEX, nitronaphthalenes, explosives (TNT, RDX, HMX), perchlorates, and anilines.
Several investigators may be viable collaborators or investment targets including:
- Shosuke Yoshida, Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology
- Aneta Urbanek, Department of Biotechnology and Food Microbiology Wroclaw University of Environmental and Life Sciences
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