The accumulation of environmental pollutants results from Anthropocene activities that expelled solvents and other harmful and hydrophobic molecules into the environment. Bioremediation utilizes microorganisms, either naturally occurring or genetically engineered, to break down environmental pollutants. Genetically engineered microorganisms, or GEMs, in theory, can digest certain pollutants. However, this does not always come to fruition. Though GEMs are economically attractive, their aptitude to do more harm than good hinders GEM efficacy. This blog post will focus on GEMs used to catabolize synthetic compounds and those not naturally found in nature.
What is Bioremediation?
Bioremediation involves manipulating bacteria or archaea in the degradation of environmental pollutants. In general, these pollutants include those that are toxic to humans and other organisms at high concentrations. Researchers can either manipulate the environment or the genetic information of microorganisms for successful bioremediation.
How is the environment manipulated for bioremediation?
The introduction of additives to the environment is a form of environmental manipulation. These additives encourage bacteria growth. For example, fertilization encourages bioremediation by enriching the contaminated soils with nitrogen and phosphorus (Crawford, R. L., & Crawford, D. L., 2005). Nitrogen and phosphorus are elements found in amino acids. Accordingly, microorganisms use these elements to synthesize amino acids that fold and create enzymes needed for healthy growth and reproduction. In simple terms, fertilization creates a minimal medium for bacteria growth and metabolic activities, including the catabolism of environmental contaminants.
The bacteria themselves may be an additive to the environment. In fact, the process of seeding introduces bacteria to the contaminated soil (Freeman et al., 2017). Hence, the bacteria added to the environment are the strains capable of catabolizing environmental pollutants.
A third method of manipulating the environment for bioremediation is “land farming.” In “land farming,” an area of soil is excavated, then is either seeded or enriched (Crawford, R. L., & Crawford, D. L., 2005). Excavation ensures that GEMs do not have direct exposure to the surrounding environment. GEM exposure to the environment has detrimental effects. After all, they pose the risk of unmanageable proliferation or transformation of genetic material into naturally occurring strains of bacteria.
How is genetic material manipulated for GEM bioremediation?
To engineer strains of bacteria, scientists must target a gene of interest. Scientists identify a target gene and engineer this gene to express a desirable enzyme needed for catalysis (Bilal, M., & Iqbal, H. M. N., 2020). Additionally, scientists must ensure the expression of only the target gene for research purposes. In simpler organisms with plasmid DNA, it is easier to manipulate DNA. Scientists may use inhibitors since associated genes lie on the same operon. There are additional methods of manipulation of bacterial DNA besides targeting specific genes.
Another method of DNA manipulation is bacterial transformation. Transformation is a process that transmits desirable microbial genes into a strain of bacteria that can be used (Bilal, M., & Iqbal, H. M. N., 2020). Of the 30,000 known strains of bacteria, researchers only use a select few to create a genetically engineered microorganism. Notably, the strains are Achromobacterm, Dehalococcoides, Pseudomonas, Burkholderia, Rhodococcusm, Comamonasm Alcaligenes, Spingononasm, and Ralstonia strains (Bilal, M., & Iqbal, H. M. N., 2020).
What molecules are catabolized during bioremediation?
During bioremediation, hazardous compounds to otherwise harmless molecules. Some of the molecules include petroleum, dioxins (a polyhalogenated aromatic hydrocarbon), polychlorinated biphenyls (PCBs), heavy metals, 1,2,3-trichloropropane (TCP), polycyclic aromatic hydrocarbons (PAHs), and 1,2 dichloroethane (DCA) (Freeman et al., 2017). Of the classes of molecules listed, most of them include chlorine, albeit.
Chlorinated compounds, such as PCBs, are abated after undergoing dechlorination. Bacteria utilize chlorine obtained from PCBs during respiration as an electron acceptor (Freeman et al., 2017). Chlorine is a great electron acceptor because of its high electronegativity value. Bacteria are unable to catabolize ever pernicious compounds, especially synthetic chemicals. The inability to digest most pernicious compounds is the reason for the creation of more GEMs. GEMs can also biodegrade chemicals, including carbamazepine sucralose, alkanes, PCBs, DCAs, and TCPs (Janssen, D. B., & Stucki, G., 2020).
The Risk of Chlorine
Chlorine is harmful to humans and animals. Contact with high chlorine levels can cause severe respiratory issues, such as pulmonary edema. In addition, coming in contact with chlorine may lead to chlorine poisoning, which varies in severity. Chlorine may also kill microorganisms that have a beneficial role in the environment.
Field Applications of Bioremediation
Researchers have underscored that there have been instances where GEM bioremediation was proven successful, but only under manipulated experimental conditions. The following are field applications of bioremediation that have had some success.
Degradation of Polycyclic Aromatic Hydrocarbons (PAHs) Using GEMs
There are polycyclic aromatic hydrocarbons (PAHs) found in coal, crude oil, and gasoline. The University of Tennessee, in collaboration with the Oak Ridge National Laboratory, conducted a field release of Pseudomonas fluorescens HK44 (manipulated strain) to degrade naphthalene, a PAH, found in the environment.
Engineering of Pseudomonas fluorescens HK44
Researchers engineered Pseudomonas fluorescens HK44 with a parent strand of genetic material harvested from manufactured gas plants contaminated with PAHs (Sayler, G. S., & Ripp, S., 2000). Scientists inserted a naphthalene catabolic into the strain, for the purpose of degrading PAHs. Insertion allowed for a lux gene fusion with the promoter for the naphthalene catabolic gene (Sayler, G. S., & Ripp, S., 2000). As a result, when P. fluorescens HK44 was exposed to naphthalene, a bioluminescent response was produced (Sayler, G. S., & Ripp, S., 2000).
Field Release of P. fluorescens
To conduct the field release of P. fluorescens HK44, scientists used an epoxy-coated lysimeter. The lysimeter was a 2.5-meter diameter canister that was 4 meters deep and 3 meters below ground (Ripp et al., 2000). Observation of the colony occurred between October 1996 and December 1999. Within the first 12 days, the colony’s population swiftly declined. But, following this period, population decline became more gradual (Ripp et al., 2000). Ripp et al. expected the population decline considering that biotic and abiotic factors may affect the system. Explicitly, biotic factors include competition and predation, and abiotic factors include temperature, pH, level of moisture, and oxygen levels. Moreover, the team found that the strain survived a lengthy 660 days post-inoculation and did not require additional energy input.
Degradation of 1,2,3-trichloropropane (TCP) Using GEMs
Groundwater bioremediation can also utilize GEMs. In a review concerning the breakdown of TCP, Janssen, and Stucki found that only genetically engineered bacterial strains can digest TCP and similar compounds, including dichloroethane (DCA). Both TCP and DCA are hydrophilic molecules. Therefore, when they interact with water, it is difficult to break the intermolecular forces between them.
Engineering of Chloroflexi
The bacteria engineered for TCP degradation was fully synthetic. Scientists gave the bacteria the name Chloroflexi. They manipulated the strain to use TCP as a growth substrate. Chloroflexi can dechlorinate TCP, forming an intermediate dichloropropanol isomer (Janssen, D. B., & Stucki, G., 2020). From there, scientists converted dichloropropanol to epichlorohydrin, a more environmentally safe compound at an alkaline pH.
Testing of Chloroflexi
Chloroflexi did not have a field release. Instead, scientists tested the bacterial strain on samples of water in the laboratory. Unfortunately, Chloroflexi produces toxic metabolites through bio-oxidation, despite some success (Janssen, D. B., & Stuck, G., 2000). They did find a safe way to degrade TCP, but this method involved extra steps. Scientists would need to extract one of the enzymes yielded in the degradation of DCA, but this requires the generation of an intermediate for degrading another compound (Janssen, D. B., & Stuck, G., 2000).
Chloroflexi’s deficit in detoxification of groundwater has prompted scientists to engineer additional strains of bacteria to serve the same purpose as Chlorofelxi. Two engineered strains, A. radiobacter, and Pseudomonas putida MC4 have an improved haloalkane dehalogenase gene. But, P. putida was the more successful strain in TCP degradation (Janssen, D. B., & Stuck, G., 2000). Scientists found maximum efficacy when P. putida cohabited the groundwater with bacteria that lack genes for hemoglobin-like proteins and flagella-encoding genes (Janssen, D. B., & Stuck, G., 2000). There are no complete field studies concerning the degradation of TCP due to wide ineffectiveness in lowering overall TCP levels without contributing to toxicity levels.
GEM Bioremediation Advantages
Success
Bioremediation holds the potential of eliminating toxic compounds from the soil or groundwater. The United States Environmental protection Agency (USEPA) cites 132 successful bioremediation activities. Of the successful bioremediation activities, 75 dealt with petroleum and petroleum-related compounds (Crawford, R. L., & Crawford, D. L., 2005).
Price
Limited success and cost-competitiveness draw many researchers to GEM bioremediation. Compared to other solutions for mending the environment, such as integrating additional chemicals, bioremediation is more cost-efficient (Sayler, G. S., & Ripp, S., 2000). Sailor and Ripp state that bioremediation costs are at least 33% less than alternatives. Alternatives include incineration and landfill methods. With time, engineering microorganisms has become cheaper because of advancements in technology.
Manipulation
Along with economic viability and some success, GEM bioremediation scientists may control the process during rate-limiting steps. Some steps in bioremediation require inputs, such as oxygen. If no oxygen is available, metabolism may not proceed. Manipulating the availability of reagents, such as nitrogen or phosphorus, can increase degradation rates (Sayler, G. S., & Ripp, S., 2000).
Convenience
Scientists and researchers need extensive equipment for monitoring progress. Genetically engineering a strain of bacteria also prevents the need for additional equipment. For example, in the lux system, the bacteria are quickly detected by computers from their bioluminescence (Sayler, G. S., & Ripp, S., 2000). As a result, bioavailability and contaminant presence was predicted from the bacteria available, quantifiable by the computer.
Strict Governmental Regulations
The United States Environmental Protection Agency has austere risk assessment diagnostics associated with the Toxic Substances Control Act. Genetically Engineered Microorganisms must comply with these diagnostics before release (Sayler, G. S., & Ripp, S., 2000). These regulations guarantee that further pollution occurs. But, the USEPA regulations hinder field releases to an extent.
GEM Bioremediation Drawbacks
Necessary Precautions
The drawbacks surrounding bioremediation using GEMs could surpass the advantages if researchers do not take the necessary precautions. Regardless of whether or not GEMs are involved, bioremediation involves extensive preliminary information, such as oxygen and nutrient concentration, microbial composition, temperature, soil particles, and even the redox potential of the soil (Crawford, R. L., & Crawford, D. L., 2005). Furthermore, bioremediation poses the risk of residual pollution. Therefore, the process requires intense monitoring, fortification of the environment, and extensive incubation times (Crawford, R. L., & Crawford, D. L., 2005).
Field Release Restrictions
When one considers all of the necessary precautions, one can find that a field release is still not entirely a field release. Confinement of bacteria colonies to a controlled and artificial system is essential for testing (Sayler, G. S., & Ripp, S., 2000). The risk of contaminating the environment surrounds the prospect of transforming genetic information into wild-type or naturally occurring bacteria. After all, legal restrictions aim to prevent this from happening.
Governmental Restrictions
Researchers must have governmental permission to study the effects of GEMs on the environment. Sayler and Ripp describe obtaining approval as a “lengthy endeavor.” In particular, in the study concerning P. fluorescens, the University of Tennesse applied for permission in July 1995 and gained it in March 1996 (Ripp et al., 2000). Even then, the field test did not begin until October of 1996, delaying the study more than a year (Ripp et al., 2000). During the process of gaining consent for the study, GEMs in a natural environment, the microbe may undergo modifications and refinements tailored by governmental guidelines. Consequently, the original microbe becomes invalid in testing.
Criticisms
One of the main criticisms of GEM bioremediation is the inability of efficacy documentation. In many cases, documenting efficacy requires chemical analysis. With this in mind, chromatography and mass spectrometry, which introduce additional chemicals to the environment (Janssen, D. B., & Stucki, G., 2020). Ethical issues surround GEM bioremediation too. Rogue microbes do hold the potential of causing further damage to the environment by releasing excess toxic metabolites. In the study by Janssen and Stucki, unwanted side reactions of chlorinated compounds may yield reactive products containing chlorine. There are technical difficulties in constructing bacteria that display the correct catabolic activities or how to utilize the target substrate in an intended way (Janssen, D. B., & Stucki, G. 2020). In addition to technical difficulties, ethical concerns surround bioremediation using GEMs. Should organisms be subject to genetic engineering to restore conditions established by anthropogenic activities?
Can Bioremediation Mend the Effects of the Anthropocene?
If researchers take the necessary precautions, they may find some success in field releases of GEMs. Field release restrictions exist to prevent disaster, and governmental constraints legally reinforce field release restrictions. Aside from these barriers, the only real issue holding GEM bioremediation back is ethical concerns. These concerns do not just surround engineering microorganisms but also concern scientists introducing additional chemicals into the environment. Despite all of these drawbacks, the success of bioremediation is promising.
GEM bioremediation has proven successful with petroleum products. Certainly, there is only a matter of time for new scientific developments to aid in the bioremediation process. With new scientific breakthroughs and technology, scientists might manipulate the genomes of microorganisms more effectively. Thus, target genes are expressed more effectively. Technological advancements and new scientific knowledge may also decrease the price of research.
Bioremediation Can Mend the Environment if Given the Chance
If society abandons stigmas and strong ethical concerns surrounding bioremediation, GEMs might be the savior of the environment. Past success has shown microorganism capabilities and has shown researchers what exactly to be wary of. Further research can find ways to prevent further damage to the environment using GEM bioremediation. Bioremediation can extend beyond the elimination of petroleum, TCPs, PCBs, and other chlorinated hydrocarbons from the environment. For example, scientists can manipulate photosynthetic bacteria to carry out photosynthesis at a higher rate. As a result, carbon dioxide, a greenhouse gas, decreases in concentration in the environment. These possibilities require ample research. But, society must decide first: are we willing to gamble our environment’s viability on the work of genetically engineered microorganisms?
Referenced Materials
Bilal, M., & Iqbal, H. M. N. (2020, June 4). Microbial bioremediation as a robust process to mitigate pollutants of environmental concern. Case Studies in Chemical and Environmental Engineering.
Crawford, R. L., & Crawford, D. L. (2005). Bioremediation: principles and applications. Cambridge University Press.
Freeman, S., Quillin, K., Allison, L. A., Black, M., Podgorski, G., Taylor, E., & Carmichael, J. (2017). 26.1. In Biological science (pp. 518–523). essay, Pearson.
Janssen, D. B., & Stucki, G. (2020, February 25). Perspectives of genetically engineered microbes for groundwater bioremediation. Environmental Science: Processes & Impacts.
Ripp, S., Nivens, D., Ahn, Y., Werner, C., Jarrell, J., Easter, J. P., Cox, C. D., Burlage, R. S., Sayler, G. S. (2000). Controlled Field Release of a Bioluminescent Genetically Engineered Microorganism for Bioremediation Process Monitoring and Control. Environmental Science & Technology.
Sayler, G. S., & Ripp, S. (2000, May 30). Field applications of genetically engineered microorganisms for bioremediation processes. Current Opinion in Biotechnology.
