Toward protein engineering for phytoremediation: possibilities and challenges

The combination of rational protein engineering and directed evolution techniques allow for the redesign of enzymes with tailored properties for use in environmental remediation. This review summarizes current molecular methods for either altering or improving protein function and highlights examples of how these methods can address bioremediation problems. Although much of the protein engineering applied to environmental clean-up employs microbial systems, there is great potential for and significant challenges to translating these approaches to plant systems for phytoremediation purposes. Protein engineering technologies combined with genomic information and metabolic engineering strategies hold promise for the design of plants and microbes to remediate organic and inorganic pollutants.     

Estimating the availability of polycyclic aromatic hydrocarbons for bioremediation of creosote contaminated soils

Bioremediation of soil contaminated by organic compounds can remove the contaminants to a large extent, but residual contamination levels may remain which are not or only slowly biodegraded. Residual levels often exceed existing clean-up guidelines and thereby limit the use of bioremediation in site clean-up. A method for estimating the expected residual levels would be a useful tool in the assessment of the feasability of bioremediation. In this study, three soil types from a creosote-contaminated field site, which had been subjected to 6 months of bioremediation in laboratory column studies, were used to characterize the residual contamination levels and assess their availability for biodegradation. The soils covered a wide range of organic carbon levels and particle size distributions. Results from the biodegradation studies were compared with desorption rate measurements and selective extractability using butanol. Residual levels of polycyclic aromatic hydrocarbons after bioremediation were found to be strongly dependent on soil type. The presence of both soil organic matter and asphaltic compounds in the soil was found to be associated with higher residual levels. Good agreement was found between the biodegradable fraction and the rapidly desorbable fraction in two of the three soils studied. Butanol extraction was found to be a useful method for roughly estimating the biodegradable fraction in the soil samples. The results indicate that both desorption and selective extraction measurements could aid the assessment of the feasability for bioremediation and identifying acceptable end-points. Received: 15 September 1999 / Received revision: 7 February 2000 / Accepted: 13 February 2000     

The use of transgenic plants in the bioremediation of soils contaminated with trace elements

The use of plants to clean-up soils contaminated with trace elements could provide a cheap and sustainable technology for bioremediation. Field trials suggested that the rate of contaminant removal using conventional plants and growth conditions is insufficient. The introduction of novel traits into high biomass plants in a transgenic approach is a promising strategy for the development of effective phytoremediation technologies. This has been exemplified by generating plants able to convert organic and ionic forms of mercury into the less toxic, volatile, elemental mercury, a trait that occurs naturally only in some bacteria and not at all in plants. The engineering of a phytoremediator plant requires the optimization of a number of processes, including trace element mobilization in the soil, uptake into the root, detoxification and allocation within the plant. A number of transgenic plants have been generated in an attempt to modify the tolerance, uptake or homeostasis of trace elements. The phenotypes of these plants provide important insights for the improvement of engineering strategies. A better understanding, both of micronutrient acquisition and homeostasis, and of the genetic, biochemical and physiological basis of metal hyperaccumulation in plants, will be of key importance for the success of phytoremediation.     

Soil initial bacterial diversity and nutrient availability determine the rate of xenobiotic biodegradation

Understanding the relative importance of soil microbial diversity, plants and nutrient management is crucial to implement an effective bioremediation approach to xenobiotics-contaminated soils. To date, knowledge on the interactive effects of soil microbiome, plant and nutrient supply on influencing biodegradation potential of soils remains limited. In this study, we evaluated the individual and interactive effects of soil initial bacterial diversity, nutrient amendments (organic and inorganic) and plant presence on the biodegradation rate of pyrene, a polycyclic aromatic hydrocarbon. Initial bacterial diversity had a strong positive impact on soil biodegradation potential, with soil harbouring higher bacterial diversity showing ~ 2 times higher degradation rates than soils with lower bacterial diversity. Both organic and inorganic nutrient amendments consistently improved the degradation rate in lower diversity soils and had negative (inorganic) to neutral (organic) effect in higher diversity soils. Interestingly, plant presence/type did not show any significant effect on the degradation rate in most of the treatments. Structural equation modelling demonstrated that initial bacterial diversity had a prominent role in driving pyrene biodegradation rates. We provide novel evidence that suggests that soil initial microbial diversity, and nutrient amendments should be explicitly considered in the design and employment of bioremediation management strategies for restoring natural habitats disturbed by organic pollutants.     

Selected factors limiting the microbial degradation of recalcitrant compounds

Summary The focus of this review is to examine some of the reasons biodegradation may not take place in the environment even though its occurrence in the laboratory has been demonstrated. Some approaches for dealing with chemical persistence will be discussed. In addition, the potential of bioremediation as an in situ clean-up technology will be considered.     

Bioremediation through microbes: systems biology and metabolic engineering approach

Today, environmental pollution is a serious problem, and bioremediation can play an important role in cleaning contaminated sites. Remediation strategies, such as chemical and physical approaches, are not enough to mitigate pollution problems because of the continuous generation of novel recalcitrant pollutants due to anthropogenic activities. Bioremediation using microbes is an eco-friendly and socially acceptable alternative to conventional remediation approaches. Many microbes with a bioremediation potential have been isolated and characterized but, in many cases, cannot completely degrade the targeted pollutant or are ineffective in situations with mixed wastes. This review envisages advances in systems biology (SB), which enables the analysis of microbial behavior at a community level under different environmental stresses. By applying a SB approach, crucial preliminary information can be obtained for metabolic engineering (ME) of microbes for their enhanced bioremediation capabilities. This review also highlights the integrated SB and ME tools and techniques for bioremediation purposes.     

Roles of Organic Molecules in Inorganic CsPbX3 Perovskite Solar Cells

Over 25% efficiencies have been achieved by organic–inorganic hybrid perovskite solar cells (PSCs). However, their practical applications are limited by the instability of the hybrid perovskite materials. Replacing hybrid perovskites with inorganic CsPbX₃ perovskites shows great promise to address the above issue and much progress has been made. To achieve high efficiency and stable inorganic CsPbX₃ PSCs, organic molecular engineering has been playing a vital role. Herein, the progress of the organic molecular engineering in inorganic CsPbX₃ PSCs is systematically reviewed. First, structure evolution induced by organic molecular engineering for inorganic CsPbX₃ perovskites is demonstrated. Then, organic molecular engineering in CsPbX₃ PSCs is categorized and reviewed (alloying in perovskite structures, as sacrificial agents, forming 2D structures, and modifying surfaces and interfaces). Finally, future research directions are suggested to further improve the performance of inorganic PSCs.     

Use of genetically engineered microorganisms (GEMs) for the bioremediation of contaminants

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.     

A Streptococcus uberis transposon mutant screen reveals a negative role for LiaR homologue in biofilm formation

Deinococcus spp are among the most radiation‐resistant micro‐organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro‐organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.     

Use of filamentous cyanobacteria for biodegradation of organic pollutants.

Biodegradation is increasingly being considered as a less expensive alternative to physical and chemical means of decomposing organic pollutants. Pathways of biodegradation have been characterized for a number of heterotrophic microorganisms, mostly soil isolates, some of which have been used for remediation of water. Because cyanobacteria are photoautotrophic and some can fix atmospheric nitrogen, their use for bioremediation of surface waters would circumvent the need to supply biodegradative heterotrophs with organic nutrients. This paper demonstrates that two filamentous cyanobacteria have a natural ability to degrade a highly chlorinated aliphatic pesticide, lindane (gamma-hexachlorocyclohexane); presents quantitative evidence that this ability can be enhanced by genetic engineering; and provides qualitative evidence that those two strains can be genetically engineered to degrade another chlorinated pollutant, 4-chlorobenzoate.     

Haloalkane dehalogenases: Biotechnological applications

Haloalkane dehalogenases (EC 3.8.1.5, HLDs) are α/β-hydrolases which act to cleave carbon-halogen bonds. Due to their unique catalytic mechanism, broad substrate specificity and high robustness, the members of this enzyme family have been employed in several practical applications: (i) biocatalytic preparation of optically pure building-blocks for organic synthesis; (ii) recycling of by-products from chemical processes; (iii) bioremediation of toxic environmental pollutants; (iv) decontamination of warfare agents; (v) biosensing of environmental pollutants; and (vi) protein tagging for cell imaging and protein analysis. This review discusses the application of HLDs in the context of the biochemical properties of individual enzymes. Further extension of HLD uses within the field of biotechnology will require currently limiting factors – such as low expression, product inhibition, insufficient enzyme selectivity, low affinity and catalytic efficiency towards selected substrates, and instability in the presence of organic co-solvents – to be overcome. We propose that strategies based on protein engineering and isolation of novel HLDs from extremophilic microorganisms may offer solutions.     

Bacterial metabolism of polycyclic aromatic hydrocarbons: strategies for bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are compounds of intense public concern due to their persistence in the environment and potentially deleterious effects on human, environmental and ecological health. The clean up of such contaminants using invasive technologies has proven to be expensive and more importantly often damaging to the natural resource properties of the soil, sediment or aquifer. Bioremediation, which exploits the metabolic potential of microbes for the clean-up of recalcitrant xenobiotic compounds, has come up as a promising alternative. Several approaches such as improvement in PAH solubilization and entry into the cell, pathway and enzyme engineering and control of enzyme expression etc. are in development but far from complete. Successful application of the microorganisms for the bioremediation of PAH-contaminated sites therefore requires a deeper understanding of the physiology, biochemistry and molecular genetics of potential catabolic pathways. In this review, we briefly summarize important strategies adopted for PAH bioremediation and discuss the potential for their improvement.     

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT

This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.     

Engineering of improved microbes and enzymes for bioremediation

Bioremediation with microorganisms is an attractive alternative to conventional techniques, such as incineration and chemical treatment, for disposing of pollutants. Recent progress in molecular biology, microbiology, and genetics is providing the driving force towards engineering improved microbes and enzymes for bioremediation. A number of genetic engineering approaches have been developed in the past several years that have proven useful in introducing/evolving the desired properties for different biodegradative pathways or enzymes. The initial excitement generated in this area should continue to pave the way for rational or irrational design of microbes or enzymes with novel remedial properties.     

HMSS2: An advanced tool for the analysis of sulphur metabolism,including organosulphur compound transformation,in genome and metagenome assemblies

The global sulphur cycle has implications for human health, climate change, biogeochemistry and bioremediation. The organosulphur compounds that participate in this cycle not only represent a vast reservoir of sulphur but are also used by prokaryotes as sources of energy and/or carbon. Closely linked to the inorganic sulphur cycle, it involves the interaction of prokaryotes, eukaryotes and chemical processes. However, ecological and evolutionary studies of the conversion of organic sulphur compounds are hampered by the poor conservation of the relevant pathways and their variation even within strains of the same species. In addition, several proteins involved in the conversion of sulphonated compounds are related to proteins involved in sulphur dissimilation or turnover of other compounds. Therefore, the enzymes involved in the metabolism of organic sulphur compounds are usually not correctly annotated in public databases. To address this challenge, we have developed HMSS2, a profiled Hidden Markov Model-based tool for rapid annotation and synteny analysis of organic and inorganic sulphur cycle proteins in prokaryotic genomes. Compared to its previous version (HMS-S-S), HMSS2 includes several new features. HMM-based annotation is now supported by nonhomology criteria and covers the metabolic pathways of important organosulphur compounds, including dimethylsulphoniopropionate, taurine, isethionate, and sulphoquinovose. In addition, the calculation speed has been increased by a factor of four and the available output formats have been extended to include iTol compatible data sets, and customized sequence FASTA files.     

Bioremediation of radioactive waste: radionuclide-microbe interactions in laboratory and field-scale studies

Given the scale of the contamination associated with 60 years of global nuclear activity, and the inherent high financial and environmental costs associated with invasive physical and chemical clean-up strategies, there is an unparalleled interest in new passive in situ bioremediation processes for sites contaminated with nuclear waste. Many of these processes rely on successfully harnessing newly discovered natural biogeochemical cycles for key radionuclides and fission products. Recent advances have been made in understanding the microbial colonization of radioactive environments and the biological basis of microbial transformations of radioactive waste in these settings.     

In situ bioremediation of monoaromatic pollutants in groundwater: a review

Monoaromatic pollutants such as benzene, toluene, ethylbenzene and mixture of xylenes are now considered as widespread contaminants of groundwater. In situ bioremediation under natural attenuation or enhanced remediation has been successfully used for removal of organic pollutants, including monoaromatic compounds, from groundwater. Results published indicate that in some sites, intrinsic bioremediation can reduce the monoaromatic compounds content of contaminated water to reach standard levels of potable water. However, engineering bioremediation is faster and more efficient. Also, studies have shown that enhanced anaerobic bioremediation can be applied for many BTEX contaminated groundwaters, as it is simple, applicable and economical.

This paper reviews microbiology and metabolism of monoaromatic biodegradation and in situ bioremediation for BTEX removal from groundwater under aerobic and anaerobic conditions. It also discusses the factors affecting and limiting bioremediation processes and interactions between monoaromatic pollutants and other compounds during the remediation processes.     

Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution

The threat of heavy metal pollution to public health and wildlife has led to an increased interest in developing systems that can remove or neutralise its toxic effects in soil, sediments and wastewater. Unlike organic contaminants, which can be degraded to harmless chemical species, heavy metals cannot be destroyed. Remediating the pollution they cause can therefore only be envisioned as their immobilisation in a non-bioavailable form, or their re-speciation into less toxic forms. While these approaches do not solve the problem altogether, they do help to protect afflicted sites from noxious effects and isolate the contaminants as a contained and sometimes recyclable residue. This review outlines the most important bacterial phenotypes and properties that are (or could be) instrumental in heavy metal bioremediation, along with what is known of their genetic and biochemical background. A variety of instances are discussed in which valuable properties already present in certain strains can be combined or improved through state-of-the-art genetic engineering. In other cases, knowledge of metal-related reactions catalysed by some bacteria allows optimisation of the desired process by altering the physicochemical conditions of the contaminated area. The combination of genetic engineering of the bacterial catalysts with judicious eco-engineering of the polluted sites will be of paramount importance in future bioremediation strategies.     

Remediation of contaminated soils by biotechnology with nanomaterials: bio-behavior,applications, and perspectives

Soil contamination caused by heavy metals and organic pollutants has drawn world-wide concern. Biotechnology has been applied for many years to the decontamination of soils polluted with organic and inorganic contaminants, and novel nanomaterials (NMs) has attracted much concern due to their high capacity for the removal/stabilization/degradation of pollutants. Recently, developing advanced biotechnology with NMs for the remediation of contaminated soils has become a hot research topic. Some researchers found that bioremediation efficiency of contaminated soils was enhanced by the addition of NMs, while others demonstrated that the toxicity of NMs to the organism negatively influenced the repair capacity of polluted soils. This paper reviews the application of biotechnology and NMs in soil remediation, and further provides a critical view of the effects of NMs on the phytoremediation and micro-remediation of contaminated soils. This review also discusses the future research needs for the combined application of biotechnology and NMs in soil remediation.     

Metal binding by bacteria from uranium mining waste piles and its technological applications

Uranium mining waste piles, heavily polluted with radionuclides and other toxic metals, are a reservoir for bacteria that have evolved special strategies to survive in these extreme environments. Understanding the mechanisms of bacterial adaptation may enable the development of novel bioremediation strategies and other technological applications. Cell isolates of Bacillus sphaericus JG-A12 from a uranium mining waste pile in Germany are able to accumulate high amounts of toxic metals such as U, Cu, Pb, Al, and Cd as well as precious metals. Some of these metals, i.e. U, Cu, Pd(II), Pt(II) and Au(III), are also bound by the highly orderd paracrystalline proteinaceous surface layer (S-layer) that envelopes the cells of this strain. These special capabilities of the cells and the S-layer proteins of B. sphaericus JG-A12 are highly interesting for the clean-up of uranium contaminated waste waters, for the recovery of precious metals from electronic wastes, and for the production of metal nanoclusters. The fabricated nanoparticles are promising for the development of novel catalysts. This work reviews the molecular biology of the S-layer of the strain JG-A12 and the S-layer dependent interactions of the bacterial cells with metals. It presents future perspectives for their application in bioremediation and nanotechnology.