Contamination of the environment is a topic oft spoken of in an incredibly broad sense. That the actions of some governments or organizations are damaging to their surroundings comes as no surprise, but discussions on the specifics and what can be done to fix the issues rarely comes up in the public societal consciousness.

The only exception is when a major event arises that is able to thrust itself into the limelight, such as when a massive oil spill or leakage develops that becomes primetime news for several days or weeks. But, inevitably, even that vanishes as it is presumed fixed and no longer a concern. And in many cases that’s true, the source of those singular incidents were corrected, but the larger issues of how they came about and how to prevent them from happening again are not and they sit in the background, waiting.

Dealing with topics the public would rather ignore is almost a de facto mission statement of science, however, as there will always end up being someone, somewhere, studying up and researching any specific field or discipline you can think of. Environmental contamination is no exception and even within that study, there is a dizzying variety of subfields devoted to different methods of treating and cleaning up contaminants in the environment.

Today, we’ll just be focusing on a single one, the one that has some of the closest ties to biotechnology due to genetics and chemistry playing such a role in how it works. The field we are discussing in this article involves using bacteria, fungi, and other organisms as natural cleanup vessels for harmful contaminants, usually by having them be consumed and converted into a new, less toxic, form. That field is known as bioremediation.

The Basics of Bioremediation

Previous efforts to clean areas of land filled with contaminants relied on chemistry to find counter components to the specific contaminant being dealt with, in the hopes of breaking it down over time. But obviously this is of limited viability, just on its face due to the complexity of biochemistry and inorganic chemistry systems. It would be expected that the breakdown pathway for many of these contaminants is more than a single step process, lengthening to the extreme the time it takes and the number of applications required to complete the process.

Therefore, environmentalists and scientists began looking to the natural world for solutions, seeing if there were organisms out there already capable of breaking down select contaminants. In some cases, they were lucky and found what they sought, often around industrial areas where natural selection had led to bacteria and the like that were capable of consuming such materials for their own energy needs. It hasn’t always been that easy, however.

Not every contaminant has an existing consumer counterpart, that we’ve found thus far at least, and even the ones we have found may not be well suited for living in the environments needed for where the contaminants are or their breakdown process may be inefficient or too slow. So genetic alteration of the available species to better suit the needs of scientists has come to be a primary part of bioremediation study. The specific techniques used though can vary considerably and they deserve some consideration.

The Techniques of Bioremediation

The process and definition of bioremediation can often be an incredibly broad understanding. It is regularly the case that the term biodegradation is treated as synonymous with bioremediation, as the end goal is to degrade any contaminant material. This can be accomplished in many ways, but the main categorization is split between in situ, treating the contaminants inside of the environments they are contaminating, and ex situ, transporting the contaminants to another location for degradation treatment.

Of course, the ability to use ex situ techniques depends on the type of contamination being dealt with, as chemicals in the soil or the water table are not as readily able to be moved somewhere else. The precise nature of the pollutant is key to choosing which technique to use, along with the type of environment, how ingrained the pollutant is, and in many cases the cost of one technique over another.

Another aspect involved with environment are things like pH of the medium that the contaminant is in, along with temperature and other abiotic factors. The variation in these features will alter the efficiency and success rate of certain degradation techniques, perhaps necessitating the use of a different method entirely if conditions are suitably unfavorable. When dealing with chemical contaminants, these characteristics are paramount to being able to activate the steps needed for breakdown into a harmless form.

Ex Situ Techniques

Due to these contaminants often being types of whole waste that are easier to transport and deal with, we’ll be starting with the list of ex situ techniques. The amount of a contaminant being dealt with plays a role in choosing to use these methods, as too large of a contaminated area can be untenable for transportation to another location for processing.

Biopiles

The most simplistic technique to be found in this category is the biopile, which is exactly what it sounds like. Contaminated dirt is taken from a polluted site and then placed into a pile at another location, usually optimally done to allow better aeration, nutrient leaching, and enhanced microbial activity, all in order to improve the efficiency of breaking down the contamination.

Piling together material to minimize surface area can also reduce the amount of volatilization of contaminants, where the material becomes gaseous and more of an aerosol. This can be extremely bad when it occurs, as it makes it easier for the contamination to spread in an environment. The biopile is also flexible toward what is needed for breaking down a specific chemical. For example, if the contaminant goes through its degradation pathway under high heat, it is possible to inject heated air into the biopile. This can also be used to increase humidity, an important factor for the growth of certain bacteria.

The downside to biopiles plays into the fact that the material is often transported to remote areas to reduce the risk of possible contamination of other environments. This remoteness means having a steady power supply is difficult to maintain. And since the use of air pumps to keep the biopile aerated to reduce volatilization is needed, this can be an issue. The use of heated air can also have its downsides depending on the bacteria being used.

But, when in a pinch and needing an area to be quickly clear of contaminants, the biopile is a definite option for bioremediation.

Windrows

Since this technique is more of an alternative application of biopiles, it will only be covered briefly here. A windrow is a pile of contaminated material that is periodically turned and mixed, similar to when soil is tilled. Water is often added during the turning process as well. This allows for the contaminants to be evenly distributed in the soil, it naturally aerates the soil without requiring air to be injected, and it increases the speed of microbial degradation of the material.

This technique is often used when dealing with hydrocarbon contaminants such as from a fuel or oil spill. Hydrocarbonoclastic bacteria are added to the soil, where they mineralize the hydrocarbons through their activity. However, windrows should be distinctly avoided as a technique when dealing with volatile contaminants prone to vaporization, as the turning of the soil makes it much easier for the chemicals to be released into the air.

Bioreactors

Quickly becoming a big part of bioremediation and already a promising look to the future for biofuels and bioenergy, bioreactors are used to convert a biological fuel source into a active fuel ready for consumption. This often requires a long series of chemical reactions that are carried out in different holding chambers within the reactor. The interior conditions of each chamber are also optimized via temperature, air flow manipulation, and what bacteria are introduced to create the most efficient breakdown of the organic material in as short a time as possible.

In this manner, rather than attempting to neutralize volatile organic compounds through other techniques, bioreactors aim to convert these components into a different, more usable form. An additional benefit is that these reactors are not limited to only soil inputs, but can also contain and convert contaminated liquids. Biostimulents and bioaugments can also be added to the mix in different chambers to help create alternative resulting compounds, as bioreactors are often used to produce multiple materials at once.

Specialized genetically modified microorganisms can play a role, if ones containing the ability to break down unique contaminants are needed. Since the bioreactors are a self-contained system, there is little risk of escape for the microorganisms, creating a perfect biosafety container.

One of the downsides to this technique is that bioreactors often need to be made for each specifical chemical and contaminant that is required to be degraded. Each bioreactor will require unique chambers for the number of breakdown steps, meaning that separate reactors will have to be made depending on what the contamination is.

Additionally, bioreactors are limited in size. If the contamination is too widespread, it will be too much for the reactors to process in any reasonable amount of time. They can still be used for secondary processing, but other techniques will have to be used for the primary volume of the degradation process. Even so, it is currently often too expensive to use bioreactors for bioremediation purposes except for the most common and widespread of environmental contaminants.

Land Farming

This final technique in this section isn’t precisely an ex situ method. It straddles the line between the exterior and interior description, where the options used are more similar to ex situ, but can often take place in the original contamination site, which would make it in situ. There is seemingly a fair amount of debate in the bioremediation community on where this technique falls.

As another simple method, land farming is quite straightforward. It requires that an area of land have the soil be compacted to make it firmer, more solid, and less permeable. Then, the contaminated soil is placed on top of it, the compacted soil acting as a base to hold up the rest. The contaminated soil that was excavated is often not moved very far when using this technique. The soil is applied carefully to spread it out, keep it aerated, and make it easier for native microorganisms to degrade the contaminant. Hydrocarbons like diesel are common contaminants that lead to this method.

Tilling and irrigation of the soil is then done to stimulate the microorganisms to breakdown the material. Nutrients usually do not need to be added to assist them in this scenario. While land farming will not get rid of the contaminants in their entirety, it will degrade the vast majority of them, leaving the rest to be less of a concern or issue to the soil microbiome.

The simple nature of this technique is what makes it so useful, as government regulation isn’t required in order to use it. The preliminary oversight of it is reduced and can be approved in short order. Basic parameters are set up on how the cleanup is to take place, what tools are to be used, and other such details. Overall though, this cleanup can be done by a much smaller group of people without requiring much machinery at all outside of the excavating vehicles.

Land farming does still have other limitations, however, including the amount of land used, the cost of excavating the soil in the first place, and the longer time frame involved in breaking down the pollutants. And, as usual by now, it doesn’t work with toxic volatile compounds prone to aerosolizing. The tilling process too easily allows for those toxic materials to be spread far and wide, so only certain contaminants are suitable for treatment with this technique.

In Situ Techniques

The flip side of the coin for bioremediation efforts are the in situ techniques, those taken at the place of pollution. Due to this locational benefit, the cost for many of these techniques are heavily reduced as compared to what is needed for ex situ methods. The success rate of these methods rely on things like porosity of the soil in order to properly break down the contaminants, among other such details.

Bioventing

The in situ counterpart to biopiles, this technique makes use of airflow to increase the activity of intrinsic microbes in the soil in the hopes of breaking down the contaminants. Nutrients and moisture are often added to create the perfect environment for microbial activity and works best on smaller spills involving petroleum products. As long as the spill isn’t too widespread, the activity of microbes should be enough to turn the petrochemicals into a harmless state. An important part to note is that too high of an airflow rate impedes the overall effectiveness of the process, so only a low, steady airflow is needed for improving breakdown speed.

The precise methods used might have to change depending on the type of soil that is polluted in order to achieve uniform oxygenation for microbes. A low air injection rate works best with clay-based soil that make it easy for air to infiltrate deep into it. But for soils suffering under permafrost, such venting is unable to be distributed properly in the hard soils and only create singular channels through it. Scientists found better results with microinjections into this kind of soil with injection rods that better allow for oxygen penetration.

Funny enough, while the primary usage is for soil aeration, the technique can also be used to deal with anaerobic bacteria. These bacteria are typically used when dealing with chlorinated pollutants and their activity is inhibited under aerated conditions. Instead of air with oxygen, the venting systems are combined with a mixture of nitrogen, carbon dioxide, and hydrogen, the latter acting as an electron donor for chlorine breakdown. For some compounds, ozone is instead used as a forceful breakdown method.

A specialized form of bioventing is known as soil vapour extraction that is focused on removing volatile organic compounds by purposeful volatilization. Due to the preferred result, high airflow rate is used with this technique and has no reliance on microbial activity, instead removing the contaminants with physical force. This, in many ways, puts this option in conflict with the rest of bioventing and often is seen as a philosophical difference in opinion among bioremediation users on which is the better choice.

Bioslurping and Biosparging

These twin techniques are variants that use bioventing processes, but in different manners.

The first technique of bioslurping combines all the specialized types of bioventing in order to indirectly apply oxygen to facilitate biodegradation. The usage relies on liquid contaminants (and some forms of soil gaseous compounds) that are then “slurped” up through a pumping mechanism. The acquisition of the pollutant products to the uptake device is thanks to partial fluidization from the oxygen flow.

The reliant part of this, however, is that there can’t be too high of a natural moisture content in the soil or it will affect airflow. Additionally, the soil must have a high enough permeability for there to be any chance for this technique to be effective.

Biosparging, on the other hand, takes more after its predecessor in bioventing by relying on microbes. Their activity is enhanced though by using air to push volatile organic compounds up to a higher soil level to better allow access by the microbial life. This should not be confused with the bioventing method of in situ air sparging that purposefully forces volatilization. The techniques are not mutually exclusive and can be used in concert for particularly recalcitrant compounds.

The focus of biosparging is on easily degraded chemical compounds like petroleum products and basic molecules like benzene. There has been evidence that the technique may be effective in treating contaminated groundwater, but it requires a proper understanding of the airflow system within the water to be able to predict where contaminants will be moved to.

Phytoremediation

The big one of the in situ options is that of phytoremediation, using the abilities of plants to deal with contamination.The types of plants used highly depend on the form of the contamination, especially if it is elemental or organic in nature.

An elemental contaminant, such as toxic heavy metals and radioactive compounds, require direct extraction via plants and sequestration away from the soil. Since the plants dying and breaking down would release the elements again, they are often collected and usually burned, with the remaining elements contained in the ash being placed in secure containment.

An organic pollutant, such as hydrocarbons or chlorine-laced molecules, require uptake, stabilization, and degradation when possible. Sometimes volatilization must be induced to remove the pollutants from the soil and drive them toward the plant roots. One of the better options is to transform the compounds into mineralized forms, actions that plants like willow and alfalfa are capable of.

When choosing the appropriate plant to use, different physical constraints must be considered, including the types of roots the plant grows and the depth to which they can reach. The parts of the plant above ground must not be accessible by animal or insect, otherwise there is a risk of transmitting the contaminants on into the food chain. The resistance of the plant to the contaminant is paramount, as the endeavor is pointless if the plant used has a low survivability to uptake of the contamination. Lastly, things like growth rate and weather conditions will affect how long the cleanup will take, with preference being given to fast-growing plants that can rapidly collect compounds in their tissues.

As one paper summarized, the procedure for plant bioremediation starts with the passive uptake process that transports material from the roots to the shoots. Then translocation and accumulation occur based on the transpiration rate of the xylem channel. This spreads the pollutants to other tissues, where they are either passively collected, chemically converted into a harmless state, or are biomineralized into an inert solid form.

It can be commonly expected that for contaminated sites that have lasted for a significant number of years, the plants able to grow significantly in the region are likely to possess some amount of phytoremediation capabilities. Plant growth-promoting rhizobacteria can be used to enhance this and often play a heavy role in phytoremediation in order to increase biomass and uptake in a shorter period of time. They can also allow for better plant tolerance to heavy metals or other negative growth contaminants that are in the soil.

An extended benefit from this technique is that, when dealing with heavy metal contamination, phytoremediation can be a good way to process and purify the collection of said metals. Then a method known as phytomining can be used to reobtain those precious metals for application in other industries. And for contaminants that aim to be converted through plant chemical processes, the growth of organic matter can prove important for soil fertility even after cleanup has finished. Due to this, it is often the case that the plants are left to grow naturally afterwards, so no additional cost is incurred from removing them.

Phytoremediation isn’t the perfect answer to all bioremediation problems, however. There are plenty of contaminant types that they are unable to breakdown or accumulate successfully. Especially when speaking about more caustic organic chemicals. They may be able to slightly process them part of the way down the required pathways, but lack the proper enzymes to truly render them inert into water and carbon dioxide, which is a common end goal with such pollutants. Since plants are autotrophic and obtain their energy from photosynthesis, they have no need for making the proteins needed for chemical synthesis and conversion of such compounds like other organisms do.

Current Examples of Bioremediation

Since we’ve finished covering all the main techniques that can be found under the aegis of bioremediation, it is now that time of the article to look to some recent publications and see what they’ve accomplished in the field. Or in the lab, we’re not picky here. The majority of these studies have gravitated to oil and petroleum-based spills, as those are a common source of soil and water contamination. But there will be a discussion on some interesting alternative topics afterwards.

Petroleum Bioremediation

When it comes to the topic of breaking down or neutralizing petroleum products, it is often a talk involving microbial usage. This is due to the ability for some microorganisms to produce biosurfactants that increase the solubility and bioavailability of the hydrophobic parts of the oil compounds that resist mixing with water. If the fluids aren’t blended together, it is difficult for the microbes to reach the material they need to degrade. In cases where organisms other than bacteria are used, biosurfactants can also be applied purposefully and directly by the people overseeing the bioremediation, though this is a more costly task.

The process of enhancing the native microbial population or adding microbes to the biome capable of breaking down petroleum compounds is referred to as bioaugmentation. It was not discussed above under techniques because it largely falls broadly under the other major methods as presented.

A study from April of 2017 looked into facilitating nitrogen-fixing bacteria (NFBs) capable of making biosurfactants and removing hydrocarbons from soil. They were introduced into a contaminated site with hydrocarbon levels at over 120,000 parts per million. An incredibly high pollutant level to deal with, the bacteria were specially chosen from species found at prior contamination sites and that had managed to thrive there.

After a year and 4 months of application in the site, the bacterial colony levels had increased from 130,000 to over a billion. Additionally, the NFBs were able to remove 80% of the hydrocarbons in that time frame, marking the first recorded scientific success of using natively found NFBs to deal with contaminants. This makes them a great candidate for future petroleum spill sites, especially in soils low in nitrogen.

In January of last year, another study considered the topic of wastewater contaminated with petroleum and the researchers prior discovery of a special strain of Bacillus cereus that is hyper-tolerant to phenol compounds and that exhibits specialized degradation pathways. They tested these bacteria in wastewater samples, splitting them into a free-roaming experimental group and a group immobilized in calcium-alginate beads, which has been claimed to improve their degradation abilities.

During a twenty day period, they analyzed the samples for things like chemical oxygen demand, which is used to tell the amount of contaminants remaining, total organic carbon, a similar reduction represents a cleaner sample, the removal of ammonium nitrogen content, and lastly an increase in biological oxygen demand. The latter shows the growth of the bacteria in the samples. That span of time was enough to allow the bacteria, especially the free cell group, to remove around 50% of the pollutants, making this new isolated strain of B. cereus to be highly effective at treating petroleum contaminated wastewater.

The next study, also from January 2017, goes a separate route from the theme of bacteria and instead targets fungi. The scientists took samples from the Mediterranean off the coast of Sicily, a region that has dealt with chronic oil spills over the years, the most recent being in 2013. Thanks to this repeated inundation with petroleum, the microscopic life in the water has had to adapt to the changing concentrations and compounds. The point of this study was to see what alterations the fungi population had come up with and if any were suitable for bioremediation purposes.

From the water samples, 67 groups of fungi were found and an additional 17 were found in the soil samples taken from the ocean floor. The species that were identified among these were the first for many to be found in an ocean environment. Then, all of the species detected were placed into test environments with crude oil as a carbon source. Among them, around a quarter were able to properly use such a compound in their biochemistry. Four of the identified strains in this smaller grouping were picked out for further experimentation. Two of those were, over the course of 30 days, shown to degrade around 30-40% of the hydrocarbon material in their surroundings, making them a good choice for future bioremediation using fungi.

As can be seen from all of these studies, there are quite a few candidates just in the past year that have come forward as viable options for treating petroleum contamination issues. Whether they will all last in the long run or whether better choices will be found or made, only time will tell. It can be guessed that biotechnology will play a role in the final decision made for a bioremediation species, as improving their uptake and breakdown speed of key chemical compounds will be a big point of future experimentation and implementation. Any reduction in time required to complete the task will benefit both the environment being cleaned and the cost of the cleanup as a whole.

Other Research In Bioremediation

This final section is a collective grab bag of other research conducted in the past year that has, I feel as the author, an interesting usage or contamination site to discuss. There are a variety of bioremediation techniques and contamination types that are too numerous to be encompassed in a single article. I will endeavor to talk about some of them here, allowing you the reader to think about further implications they might have on the future of bioremediation.

The first study to look at comes very recently from a South African journal and published in December of last year. It features a problem that is of particular concern to the country in question due to the widespread mining of resources that has been done throughout the land over prior centuries. After a mine has been exhausted, that doesn’t mean all the metals in the surrounding earth are gone, just that it isn’t cost-effective to try and dig them up.

However, this can create a major problem. Over time, sulfide chemicals in the soil break down due to exposure to oxygen in the mine tunnels. This, in turn, promotes the leaching of heavy metals into the same open space. If there is liquid anywhere in the tunnels, which is likely, then the result can be a heavily acidic and corrosive wastewater solution. The process of its creation can also be exacerbated by certain solutions and contaminants used by miners for digging and that have been left behind.

The acid liquid drainage can percolate up to the surface, spilling out of the mine or leaking out through the soil above the mine, damaging the surrounding environment and turning the land toxic for life. Thus, the ability to use bioremediation to clean up these polluted liquids is paramount in not just South Africa, but throughout the continent and elsewhere. To deal with this problem, the researchers chose algae as the prospective contender due to previous studies showing success in treating acid mine drainage, especially with heavy metals involved.

Their review of the topic found that, while algae usage for the dilemma has been common since the 1990’s, there is room for improvement. They are able to deal with both organic and inorganic contaminants, but their degradation process is fairly slow and their nutrient levels need to be closely monitored so as not to kill them off. The authors of the study hope that future genetic engineering tinkering may be able to fix a lot of these drawbacks and make algae an even better option to deal with wastewater from abandoned mines.

The consequent matter of conversation is about a study published in September that looked at melanin producing microorganisms for the purposes of not just bioremediation, but also protection from radioactive emissions (radioprotection). To begin, melanins are incredibly complex biomolecular structures, produced as pigmentation that are able to absorb electromagnetic radiation and adsorb radioactive particles and many chemicals. The pigments, by themselves, are often not ordered into the secondary planar structures necessary to exhibit these properties, but it is only when actively expressed by living organisms that this complexity comes to the forefront.

The melanin-forming bacterial species already known and recorded by science live in extreme environments around the planet, including the arctic north, petroleum polluted land, and radioactive soil like the region around Chernobyl. The ability to use microbes as an on the go source of bioremediation for chemical buildup and as potential radiation blockage may be of high importance in the future, especially in space travel and colonization of other worlds. If melanin producers can manage that necessity, then they seem like one of the best options for the practice. Since the degradation of radioactive particles turns the excess energy into heat, there can also be uses found for such a byproduct.

The last of the extended alternative topic studies entails a discussion on bioremediation of food products, something we haven’t touched on as of yet. When it comes to foodstuffs like maize, there is one primary contaminant that is constantly worried about, fungal infections that include a release of aflatoxins. These toxic compounds are extremely dangerous and can stunt growth and promote cancer formation. Even a single confirmed piece of contaminated corn is enough to throw out the entire shipment, wasting thousands of pounds of food.

Thus, research into finding ways of both blocking infection in the first place and also removing aflatoxin contamination after it occurs is a common object of study in agricultural fields. A study back in August by Italian scientists thought to use another fungus, the king oyster mushroom, to combat aflatoxins. This mushroom is almost a household food product in some places, being an edible delicacy. When the researchers grew the mushroom in a nutrient rich broth with aflatoxins at a concentration of 500 nanograms per milliliter, they confirmed its ability to reduce the contaminants by up to 99% within 10 days and more or less 100% by 30 days.

Of course, this was under the most optimal growth conditions in a friendly environment. When applied to a solid agar medium with the same aflatoxin inclusion, the growth of the mushroom was significantly inhibited. But the researchers found that by adding 5% wheat straw to the medium, the mushroom was able to resist the aflatoxin better with no observed inhibition. Another supplementation test optimized things further by also adding 2.5% maize flour to the medium, leading to 71-94% degradation of the aflatoxins after 30 days.

The concluding experiment directly exposed king oyster mushroom spores to corn infected with aflatoxins, with the same supplementary nutrients added. After 28 days, the mushroom was confirmed to have removed 86% of the contaminants with no meaningful change in mushroom yield or its biological efficiency. The mushrooms were also directly sampled afterwards to make sure the aflatoxins hadn’t just been transferred into their tissues. But no evidence of that was found, indicating that degradation and breakdown of the toxins had indeed been what happened.

Therefore, for the purposes of animal feed production, king oyster mushrooms may be a good candidate for cleaning up aflatoxin contamination of corn and saving pounds and pounds of food from being thrown away.

The Cleanup

That covers most of the simple side of bioremediation, but it isn’t comprehensive in the least. This scientific field is a complex one and it crosses lines into most other fields of study as well, using organisms from across the biological spectrum. There is no one correct way to deal with environmental pollutants and the answer is often a combination of techniques. Not just those found in bioremediation, but also combined with other forms of organic chemistry and biotechnology. In many ways, each contamination site is unique and must have a unique response to deal with it.

We work to create generalized solutions for these problems and we are making clear progress toward cleaning them up. But there is still a long journey ahead and research to be done. As new forms of power generation, medical enhancements, and more are developed, there are new concerns to be dealt with and likely new types of contamination that we will have to find ways to fix.

Bioremediation is an ongoing and evolving field with much to provide for the environment and for our future endeavors beyond that as a species. We may not be able to prevent accidents from happening all the time, but with this technology, we can at least clean it up after the fact.

References

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Photo CCs: Restoring an urban stream (after) (7557277790) from Wikimedia Commons

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