INTRODUCTION

Bio-remediation is the act of using natural life (bio) to break down and remediate a contaminated site, surface or object.  Restoring a surface or object to a condition which is no longer hazardous to the environment, is the prime objective of this process. 

Using microorganisms and special enhancement solutions to break down the hydrocarbon pollutant is the newest and most successful technology being utilized in this process.  There are many different methods accepted in these processes.  However, biodegration is the preferred system in most cases.  The related system can be categorized as follows:  

BIODEGRATION:  The degrading or chemical breakdown of a substance or compound that’s using living microorganisms.  

BIORESTORATION:  The restoring of an object or site to a biologically suitable condition similar to it’s original state by the utilization of natural microorganisms.  Nature in itself acts as a built in check and balance system.  Within time (years), nature will usually create a defensive mechanism (microbial) to re-establish a natural balance.  

BIOSTIMULATION:  The act of physically manipulating a contaminated site to enhance the growth of existing or added microorganisms.  Nature in time will populate an area with microorganisms to start a natural remediation process.  This process is drastically enhanced during properly engineered biostimulation.  

BIOAUGMENTATION:  This is the utilization of specific living forms to enhance or augment the natural processes taking place.   
The major benefit of bio-remediation is transformation and not transportation.  The cost of transformation is lower and requires little or no specialized equipment.  Bio-remediation is the newest technology in remediating hydrocarbon contamination in an environmentally conscious world.  

Bioremediation is a combination of biodegration, biorestoration, biostimulation and bioaugmentation.  The results are faster and more efficient remediation  than other methods.   This enhanced approach can achieve dramatic results in relatively short periods of time.  

HOW DOES IT WORK? 

Throughout the past twenty-five years, many technologies have been introduced to help with the remediation of petroleum contaminated soils.

The major benefit of bio-remediation is transformation and not transportation.  The cost of transformation is lower and requires little or no specialized equipment.  Bio-remediation is the newest technology in treatment of hydrocarbon contamination in an environmentally conscious world.
 
Bioremediation is a combination of biodegration, biorestoration, biostimulation and bioaugmentation.  The results are faster and more efficient remediation than other methods.   This enhanced approach can achieve dramatic results in relatively short periods of time.
   
The first stage of this technology includes the introduction of a proper mix of “activated” peat moss and nitrates to target the contaminates.  After initial application and tilling, the process now requires monitoring and repeated tilling and/or watering, depending on the weather conditions.  The “activated” peat, has been dried to create voides in the capillaries which will only absorb hydrocarbons.  Once encapsulated, acceleration of the breakdown of the hydrocarbon is increased in part due to the added surface area and exposure to oxygen of the hydrocarbons.
   
The activated peat technology was researched by the Federal Government (FORSCOM) using a bio-cell (landfarming) approach with the omission of repeated watering and tilling.  The study showed that the activated peat technology reduced TPH-GRO contaminations from 615 ppm to 10 ppm (81%), within 84 days.  Similarly, the “no frills” treatment reduced TPH-DRO contaminations on a second site from 494 ppm to 56 ppm in the same amount of time.   The purpose of this study was to research and find bioremediation methods that would dramatically reduce their costs.  The comparative costs, including the construction of two biocells have been determined to result in an annual savings of 60% over soil safe or chem waste disposalalternatives (from $209,900 or $242,000 to an annual cost of $66,800).  Permanent implementation of this technology has been approved for this Federal Facility to begin in 2002.
 
Bio-Matrix provides optimum absorption of the hydrocarbons in its capillaries, while preserving the natural occuring bacteria in the soil.

…Why soil bacteria that clean up metal pollution find it so hard to let go.

31 May 2000 
by:  DAVID ADAM

Bacteria wearing sticky protein coats could soon become front-line soldiers in the battle to clean up polluted soil. Bugs covered with metal-binding proteins can mop up pollution and boost plant growth, new research shows.

The cell surfaces of many enzymes latch onto heavy metals, but this metal 'sequestering' is too feeble to significantly reduce metal pollution in soil. Bugs can be made stickier by modifying them so that they produce cadmium-binding mouse proteins, Victor de Lorenzo of the Centro Nacional de Biotecnología in Madrid, Spain and his colleagues now report. Although the toxic metal remains in the soil, once bound to the modified bacteria it is less likely to be taken up by plants and animals.

The problems of heavy metal pollution are well known in Spain. In April 1998, about five million cubic metres of polluted water spilled from a mine residue pool near Seville, ruining crops and leaving surrounding farmland useless. Although the floodwater was successfully diverted away from a nearby national park, the ecological effects are expected to linger for years, as the zinc, lead and other metals left in local groundwater work their way through the food chain.

Microorganisms offer a cheap way to clean up pollution. Bugs that convert toxic substances, including oils and solvents, to harmless gas are becoming increasingly popular, not least because they are robust, easy to manage and don't stop working to read the newspaper. But they are not alchemists -- metal pollutants stay metal, whatever bacteria do.

But 'treating' metal pollution does not necessarily mean removing it from soil, de Lorenzo's team shows. They can make cadmium in soil less harmful by attaching it to the bacteria Ralstonia eutropha, a strain naturally resistant to heavy metal pollution.

They do this using 'metallothioneins': proteins produced by animals including mice that have an affinity for heavy metals. Few microrganisms produce metallothioneins, but de Lorenzo and his colleagues find that R. eutropha can be persuaded to do so -- by giving it the mouse gene.

These genetically modified bacteria escape many of the strict conditions governing other GM experiments. "There are regulations, but they are far more simple than those applied to GM plants," de Lorenzo says. "None of the engineered genes is this case can pose any imaginable risk, even in the very unlikely case that other enzymes capture them," he adds. The researchers also give the soil bacteria part of another gene that encourages the cadmium-luring proteins to sit on the bacterial outer membrane, so that they are fully accesible to the pollutant.

In tests using cadmium-contaminated soil and tobacco plants, the modified bacteria bind three times as much cadmium as unaltered bugs, reducing the cadmium's toxic affects on the plants. Over four times as many plants grow in 'treated' as in 'untreated' soil, but plant growth is still almost 90 per cent lower than in uncontaminated ground, the researchers report in Nature Biotechnology1.

The results are "promising, but not spectacular," says Derek Lovley, a microbiologist at the University of Massachusetts who specializes in bacterial pollution treatment. "Further improvements will be required before inoculation of soil with these organisms can be considered an effective remediation strategy."

But the study does prove that the technique could work, making further work in this area crucial. As Lovley says: "there are currently so few other practical options for effective, yet inexpensive remediation of metal-contaminated environments."

References  Valls, M., Atrian, S., de Lorenzo, V. & Fernández, L. A. Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil. Nature Biotechnology 18, 661 - 665 (2000). © Nature News Service / Macmillan Magazines Ltd 2001 http://www.nature.com/nsu/000601/000601-6.html

Digging deeply

Subsurface science unearths previously unknown organisms  that could be a gold mine for environmental remediation.
 
Fascinating creatures are at work deep in the earthþand they do interesting things. Environmental Sciences Division researcher Tommy Phelps has encountered them during the past several years in his work with the DOE Subsurface Science program, which has investigated the techniques for drilling holes deep in the earth and the problems that arise when you dig way down.

What Phelps and fellow researchers have found in the deep subsurface sediments are thermophilic bacteria—that means they like it hot—that have the capability to alter metals to produce magnetite, or magnetic iron. Some researchers are interested in them from an evolutionary standpoint. However, because these bacteria reduce metal compounds in an electron-swapping process similar to our breathing, they could be useful in waste remediation technologies as well as other applications.

Phelps touts these subterraneous microorganisms because of their comparative efficiency in producing their magnetic leavings."We haven't found any microorganism that doesn't have a cousin on the surface that can do the same thing, but the thermophilic bacteria can do it better, faster, and under more extreme conditions. We believe this capability can be applied to a variety of problems, the most obvious being mixed waste treatment."

Environmental remediation was a major reason DOE continued to study deep drilling when other agencies' incentive to dig ended at around 100 meters. Because, for instance, plumes of the solvent trichloroethyleneþa notorious pollutantþcan sink more than 100 meters deep and expand for several kilometers, shallow holes don't address the DOE researchers' questions. "When you dig deeper than 100 meters, the costs go up and so do the problems," Phelps said."Deep holes take an entirely different set of technologies and expertise. It is also expensive—you have to allocate your resources to where you dig one hole every two or three years."

The thermophilic bacteria were first encountered at an oil and gas deposit exploration site near Fredericksburg, Virginia. Researchers discovered the magnetite-producing bacteria at a depth of about 1000 meters. The site lay in the Taylorsville Triassic Rift Basin, a region with an underground layer of sediment that was formed 200 million years ago during the Triassic Period, then covered by tectonic activity. The ancient layer is effectively sealed off from the surface."It is geologically and hydrologically isolated from surface effects," Phelps said."It has groundwater that is probably 100 to 150 million years old."

That insulating layer has eroded over time enough to make it accessible, although not easily accessible. To support the 76-meter drilling rig and be able to truck in supplies in rainy weather, the oil and drilling companies literally floored a 1.5-acre tract of land with two to three layers of oak planking. It took 67 semi trucks to haul in the equipment. "The DOE assistant secretary at the time came out to visit. He was intrigued by the scope of the project—the hole went to a depth of 2.3 kilometers—and the industry- laboratory-DOE collaboration," Phelps said. A similar hole in the loftier terrain of the Piceance Basin near Grand Junction, Colorado, produced similar bacteria, although the two regional rock formations were formed at different times and are far apart."We started looking for new organisms, and found some," the ESD researcher said."We found organisms that reduced every metal that we threw at them—iron, manganese, chromium, cobalt, and uranium.

These scanning electron micrographs show two bacteria cultures with externally deposited magnetic oxides.

These microorganisms use metals as electron acceptors, like we use oxygen. Just like we make carbon dioxide from oxygen, they take iron and make magnetite and maghemite—magnetic minerals." Phelps is currently working with two postdocs, Chuanlun Zhang, a geoscientist, and Shi Liu, a microbiologist specializing in anaerobic organisms. They are working with the Chemical and Analytical Sciences Division to explore the potential of the enzymes."CASD has been immensely helpful in the basic sciences avenue, and Zhang's background in geoscience has helped us in collaborating with CASD's Dave Cole."

Phelps notes a good deal of interest in these netherworld enzymes in a number of venues."An oil industry rep said that where he finds magnetite, he finds natural gas," he said. Other scientists want to study the isolated bacteria's
respiratory systems on an evolutionary basis—thermophilic iron reduction as a possible precursor to oxygen respiration.

One speculative application could be rheomagnetic fluids, in which a fluid like oil with magnetic particles added can be rendered solid by applying an electric current. Other uses could lie in the production of high-speed lubricants, paints, photographic films, and even magnetic tracers for diagnostics. The real interest is in treating mixed waste, including metals and solvents."All waste, really," Phelps said. Phelps theorizes that these bacteria could be introduced into water polluted with heavy metals. Once the organisms have reduced the pollutants into insoluble metal compounds, retrieving it could involve a simple separation process.

And, if their environment can be heated enough, thermophilic bacteria could be very efficient at cleaning up groundwater polluted with solvents, Phelps said.

A patented Westinghouse technology that arose from the early stages of the DOE Subsurface Science program, PHOSter, received an R&D 100 award. The phosphate- removing technology is now used in seven states. The subsurface program, however has ended, and its biotechnological applications have been rolled into the Natural and Accelerated Bioremediation program. The thermophilic bacteria studies are currently in funding limbo. That hasn't kept news from getting out about the weird bugs underground. Recently, a Web page about unidentified flying objects made mention of the Subsurface Science Program and its work with "bacillus infernus,"— bacteria from hell—and offered a link to an article on the subject.

It's fairly safe to say that drilling a hole to these depths represents a journey into the unknown. Any suggestion, however, that these underground bacteria might be long-sequestered alien life forms leaves Phelps in a merry state.

http://www.ornl.gov/publications/labnotes/aug96/phelps.htm

Bioremediation: Bacteria Do Our Dirty Work
By Douglas Page 

In August of 1992, 133,000 liters (35,000 gallons) of Jet A aircraft fuel were discovered to have somehow seeped out of the pipeline and underground storage tanks into two areas of soil beneath Van Nuys Airport in Van Nuys, California.

By toxic spill standards, this wasn't headline news, but it was big enough to draw the attention of environmental agencies that feared the fuel could reach ground water. One gallon of contaminant can pollute 300 million gallons of ground water. The two soil areas contained elevated petroleum hydrocarbon concentrations of 24,000 parts per million (measured in total petroleum hydrocarbon, or TPH, units), three times the level that requires remediation. The combined plumes soaked an area some 150 by 90 feet, to a depth of 90 feet. At discovery, the plume had reached one-third of the way to ground water.

Traditional cleaning methods for the site (located adjacent to a major runway) could have disrupted airport activity for three to five years. These traditional methods involve applying caustic, solvent-based cleaners or excavating and hauling. Caustic chemicals often create even more environmental problems, and excavation essentially just sweeps the contamination under a different rug.

Instead, the airport tenant chose a proposal from Biotreatment, Inc., San Diego, which used a technology called bioremediation that cost less, took less time, and did not disrupt airport activities to any extensive degree. Within 90 days, in situ bioremediation reduced TPH levels by an average of 80 percent. Monitoring at that time showed the degradation rate had leveled off at 2,000 to 2,500 ppm--75 percent below action levels.
 
How Bioremediation Works

Basically, bioremediation harnesses the ability of microorganisms (such as bacteria or fungi) to remove pollutants from the environment. In its natural state, this process is called biodegradation. Biodegradation is as old as life itself.

When humans enter the picture by manipulating conditions, the process is called bioremediation.

Bioremediation is the natural way of reducing toxic organic materials to harmless carbon dioxide, water, and various forms of salt. It is, in fact, the same process that takes place when grass clippings and garden waste are composted for later use as a soil nutrient for future plantings. Enzymes existing naturally in all soil and water produce enzymes that break down hydrocarbons into smaller, less toxic materials.

Using modern methods, science has found ways to accelerate and improve the effectiveness of biodegradation. Naturally occurring enzymes identified and isolated for their ability to degrade specific hydrocarbon contaminants such as oil and gasoline are now being cloned and applied in industrial strength to hazardous waste sites. The enzymes, when combined with nutrients, pH stabilizers, oxygen, and surfactants (detergents), yield a product that, when introduced into contaminated soil or water, optimizes the environment so bioremediation can take place.

Bioremediation is currently being used to manage municipal sewage, clean up oil spills, remediate ground water contaminated by underground storage leaks, treat industrial waste water, and reclaim a variety of hazardous waste sites.

Biotechnology firms such as Advanced BioTech, Visalia, California, market naturally occurring microorganisms packaged in a dry, dormant state. BioTech's hydrocarbon-digesting enzymes, for instance, are sold in one-half pound (227 gram) or 2.5 pound (1.1 kilogram) containers, including specially formulated biochemical nutrients--a concoction well suited to remediate benzene, amines, phenols, cresols, naphthalene, alcohols, petroleum hydrocarbons, and pesticides from refinery and petrochemical waste sites.

Once bioremediation treatment commences, the hazard is swiftly and dramatically reduced, neutralized, or eliminated through mineralization--a botanical term for the decomposition of organic matter in soils by microorganisms, which releases mineral elements (nitrogen, phosphorus, potassium, sulphur) as inorganic ions. Altogether, more than 70 different enzymes are known to be capable of degrading petroleum components. The technology is approved by the U.S. Environmental Protection Agency and the EPA's Canadian compliment, Environment Canada, as well as other regulatory bodies worldwide.

The bugs have appetites for more than petroleum byproducts. Researchers are using genetically engineered fungi, bacteria, and algae as "biosorption" systems to capture polluting metals and radionuclides, including mercury, copper, cadmium, and cobalt. One company, The Institute for Genomic Research (TIGR), has successfully sequenced a microbe that can absorb large amounts of radioactivity. TIGR scientists hope to use the genes that code for "uranium-gobbling" to fashion new biological means of cleaning up radioactive dump sites.
 
Some of the early work in the microbial technology behind bioremediation was conducted in the 1950s by a scientist named Howard Worne, who was commissioned by the U.S. Army during the Korean conflict to develop a new generation of military fatigues that would not rot in the moist, humid Asian climate. In the course of his work, Worne uncovered a microorganism that broke down fabrics that were previously thought to be non-biodegradable. From this discovery, Worne speculated other microorganisms might exist that could degrade other materials. Eventually, Worne isolated an organism that was capable of degrading phenol (carbolic acid), a common organic pollutant. The bioremediation industry took root in Worne's work.
 
Bioremediation Today

Biotechnology has emerged has an industry with one of the highest growth potentials for the twenty-first century. After just 10 years in the biotechnology revolution, there are already more than 1,300 biotechnology companies in the U.S., with a total of nearly $13 billion in annual revenue and more than 100,000 employees. With more than 200 million tons of hazardous materials being generated annually in the U.S. alone, the cost of cleaning up toxic waste sites is now estimated to be in excess of $1.7 trillion. Bioremediation, a practical and cost-effective method of removing hydrocarbons from contaminated areas, could do a lot of the dirty work for us.

Bioremediation, however, is not a panacea for soil and ground water contamination. There are limits to bioremediation's effectiveness. Microbe growth is inhibited by heavy metal concentrations, making bioremediation unsuitable for some cleanup efforts. Plus, not every chemical has an appropriate bacterium, and when genetically engineered enzymes are used, this constitutes release of a new organism with unknown consequences. Employing bioremediation is not as simple as just pouring a microbe soup over a spill site. Many smaller environmental firms have stopped doing bioremediation because they have realized there is much more to bioremediation than mixing a few nutrients and water with soil and then seeding with a few grams of the common soil bacteria Pseudomonas aeruginosa. Bioremediation, like the rest of the environmental industry, is maturing. Regulators and clients have gotten smarter. Bioremediation is now a technically viable remedial option based on good science and engineering.
Successful, cost-effective bioremediation programs are dependent on hydrogeologic conditions, contaminant signature, microbial ecology, and other factors. Biotreatability studies are necessary to evaluate site conditions, including such analyses as:

Screening studies to obtain biodegradation parameters such as electron acceptors/donors, oxidation-reduction potential, and pH (a logarithmic index for hydrogen ion concentration)

Microbiological assays to determine microbial growth conditions, degrader population densities, and presence of enzymes capable of destroying contaminants of concern

Microcosm studies to evaluate bioremediation potential under controlled conditions

The preliminary analysis is performed by experts in the field--environmental engineers, soil scientists, hydrogeologists, and chemical engineers. Indeed, this may be the one place where chemical engineers can apply their skills to the remediation, instead of the creation, of toxins.

After the biotreatability assessments are complete, those involved can decide whether to apply intrinsic (passive) bioremediation or enhanced (engineered) bioremediation.

Intrinsic bioremediation of toxic organic compounds is accomplished by using indigenous microorganisms, principally heterotrophic (carbon-requiring) bacteria, which transform the contaminant into an innocuous byproduct. In this simplest form of bioremediation, contaminated soil is "bio-stimulated" with nutrients and an oxygen source, like peroxide, to encourage the proliferation of existing enzymes, which in turn degrade the contaminant more rapidly. When the nutrients and contaminants are depleted, the organisms are left to return to their original levels or are removed via a filtration process.

In other cases where risk to the water table is more immediate, enhanced bioremediation may be necessary. This engineered form of bioremediation can be performed in situ through biosparging (spraying), bioventing, or hydrogen peroxide/inorganic nutrient supplementation. In these cases, the contaminated soil is often tilled into uncontaminated surface soil along with the nutrients that will stimulate indigenous bacteria to degrade the contaminants. In some cases the soil may be aerated or flushed with liquids to assist in the removal of pollutants. The duration of the process varies from a few weeks to several months.