Bioaugmentation: Sending in the special troops

• Nov 01, 1999

By adding certain materials to a contaminated environment, scientists can speed up the rate of natural degradation. This process, generally referred to as bioremediation, has been splintered into different factions and techniques, depending upon what "materials" are added to the site. This usually comes down to biostimulation versus bioaugmentation.

The advocates of biostimulation believe that since microbes are ubiquitous, the indigenous microbes at the contaminated site will take care of the pollution and all that is necessary is the addition of fertilizers, nutrients and possibly special chemical compounds to speed up the growth of that indigenous microbial population. On the other hand, bioaugmentation proponents believe that the way to clean up the pollution is to inoculate the site with a consortia of specific contaminant targeted microbes in high densities. In both techniques, the environment must be carefully controlled and monitored for optimal microbial growth, and some engineering of the site may be necessary.

A numbers game
Whichever approach is used, biostimulation or bioaugmentation, there is one unalterable fact. In order to have any significant hyperdegradation occur, there must be a minimum of 1 million oil-degrading microbes per gram of contaminant to override the indigenous population of microbes. Anything less and the bioremediation process becomes a very slow one, if it works at all. So it all boils down to a numbers game. The more hydrocarbon-degrading microbes present at the site of the spill, the faster and more efficient the remediation process.

Although microbes are generally ubiquitous, only a small percentage, usually less than 1 percent, of the indigenous microbes may have hydrocarbon-degrading enzymes. If the proper microbes are not present, the site remediation will be delayed, evidenced by the persistence of long-term pollution. Microbes may adapt, over lengthy periods of time, using the most prominent energy source available. Therefore, if a petroleum hydrocarbon spill happens at a site that has seen numerous similar spills over a period of years, there may well be a population of petroleum hydrocarbon-degrading microbes present. Conversely, if the accidental release occurs at a site that has not experienced numerous petroleum hydrocarbon spills, there may not be a suitable microbial population. In either case, the biostimulation method of adding fertilizers, nutrients and special chemical compounds may or may not be effective.

The microbial elite
In contrast to biostimulation's use of indigenous microorganisms, the process of bioaugmentation involves inoculating a contaminated site with a proven, highly concentrated consortia of hearty hydrocarbon-degrading microbes. The only lab test needed is a simple, quick and inexpensive biocompatibility test to determine if there are any substances present that would be toxic to the microbes, thus preventing them from doing their job. Once the site is inoculated, the microbes quickly adapt to their new environment and begin the breakdown of the pollutant. The speed with which this occurs is usually relative to the density of the needed microbe population.

Oppenheimer Biotechnology Inc (OBI) Formula 1^TM, is one example of an effective bioaugmentation tool that uses microrganisms specifically selected for their ability to ingest petroleum hydrocarbons. The microbial process is relatively constant, but the application methodology is dictated by the geometry, geology, geochemistry, water and oxygen, as well as the basic requirements of substrate and nutrient contact at the cellular level. Unfortunately, the need for the protection of intellectual property does not permit fine details of the process to be presented.

Generally the product is applied at a density of 1,000,000 viable cells per gram of water or soil. This has been successful to dominate the normal soil microflora. Nutrients including essential trace elements are supplied relative to the concentration of hydrocarbon-carbon. When needed, oxygen is made available through a specialized biocatalyst or an inorganic oxygen donor.

Triumph over TCE
The use of the Formula 1 product resulted in the successful closure of a trichloroethylene (TCE) contaminated site in Georgia. This site was under the buildings of an old auto parts manufacturing plant where TCE had been used to wash the parts. The TCE had been disposed of in a well that dropped the material onto the ground under the building. One small area had been removed, and the contaminated soil had been excavated so a profile of approximately 10 feet was exposed. Because the building covered the site, and the soil absorption, the decision was made to inoculate in situ by high pressure.

A site analysis had been made to identify the plume under the building. The known contaminated area was 1,100 cubic meters (m₃). The TCE content of the soil ranged from 3.766 parts per million (ppm) to 0.682 ppm, indicating the absence of a dense nonaqueous phase liquid (DNAPL) zone. The soil was a fine clay-sand with a high absorption. Percolation tests with water indicated a penetration of 1 centimeter per minute. This information was used to determine the injection point grid pattern. The soil was injected horizontally and into the floor of the existing trench. Microorganisms, nutrients and a biocatalyst were injected into the soil at a pressure of 3,500 pounds per square inch (psi), to the depth of contamination. This high pressure does not affect the microorganisms. The injection grid was set to distribute approximately 1 million cells per square centimeter throughout the contaminated zone.

Bioremediation was implemented March 23 to 26, 1998. A single injection pattern was completed.

TCE concentrations between 0.682 ppm and 3.766 ppm in the soil were reduced to acceptable levels, in accordance with Georgia's Environmental Protection Division's Hazardous Response Program Guidelines for Type 1 and 3 soils. The laboratory results reported values for TCE between below detection limit to a maximum of 0.23 ppm, proving the effectiveness of bioremediation.

The bioremediation treatment proved to be a cost-effective method that saved almost $1 million by avoiding the removal of buildings and soil for disposal.

Tackling PCBs
This bioaugmentation product has also been successful in cleaning up a site located in Delmas, South Africa, that was contaminated with polychlorinated biphenyl (PCBs). The site was a relay station on which the transformer leaked approximately 50,000 liters of transformer oil containing PCBs (primarily Askarel 1242) to the surrounding soil of fine sand and clay. The contaminated area was 36 by 20 by 1 meters. The average hydrocarbon levels (10 sample areas) were at 150,000 ppm and PCB levels at 412 ppm.

The cleanup started on August 8, 1996. The site was treated with OBI's BioZorb^TM bioaugmention product at a concentration required to inoculate the contaminated soil at 1 million viable cells per gram of contaminant. An equivalent weight of nutrients containing nitrogen, phosphorous and potassium, plus trace elements required for the biodegradation of the pollutants, added in water to provide 10 percent soil moisture. Three treatments were applied during the first six months.

Conclusions
Funds were not available for comparative controls to be established in connection with the above-referenced projects. The data indicates that the sites were cleaned up by the process to acceptable levels; as the microorganisms continue to survive and decompose, the process of recycling will return the areas to background levels. Intrinsic bioremediation may have returned the sites to background, but experience has shown that this is a lengthy process compared to applied bioremediation, and in the interim the areas remain contaminated.

Generally, intrinsic bioremediation requires a study of the background microorganisms, the addition of nutrients and oxygen sources, extensive mixing, and continuous sampling. The time factor to return to background levels cannot be estimated until an adequate decline curve is generated.

Data based on other international projects using this bioaugmentation technique have shown that the addition of appropriate microorganisms will eliminate indigenous microbial estimation, reduce continuous sampling and reduce the time factor to a reasonable period, depending on the type and concentration of the pollutant and other environmental conditions. The initial mixing effort and cost of nutrients will remain approximately the same as for intrinsic bioremediation.

New frontiers in the nanoworld

Since the designation of the new bacterial domain Archaea, scientific research indicates that an entire new ecosystem of unknown microorganisms has been and is active in our environment, as described in Brock et.al. Biology of Microorganisms, 1994, Prentice Hall, and Sieburth, Sea Microbes, 1975, Univ. Park Press, Baltimore.

Scientists are describing this vast new ecosystem of unknowns as the nanoworld. Everywhere, even potentially on Mars, scientists are discovering these new organisms, from the microbial life in deep sea vents, to ice cores and water under the ice at Antarctica, to hot springs.

All point to a new frontier in the field of microbiology. The result for bioremediation has been the acceleration of the natural cycles of composting polluted soil and water.

While approximately 27,000 different bacteria and strains have been identified, the Archaea and Procaryote must have a million or more different forms. This is deduced from the requirement for microbes to recycle the approximately 7 million organic molecules and their complexes that have evolved to form the structure of the diverse present day life forms on earth. How many Prokaryote share the planet with us? According to William Whitman, a microbiologist at University of Georgia, the number is 5 x 10³⁰. If one estimates that the Archaea have an equivalent mass, their significance to the ecosystem must be highly relevant.

The Procaryote microbial biomass is equivalent to 5 trillion tons or cubic meters. In the publication, Productivity of the World's Ecosystems, National Academy of Sciences, 1975, annual primary productivity was estimated at 2.35 X 10¹¹ metric tons or approximately the same as Whitman's estimations concerning Procaryote biomass. Other estimates indicate that consumer biomass is 1 percent of annual plant productivity. Unfortunately, the bulk of the Procaryote and Archaeal bacterial biomass that is responsible for recycling has been little studied.

Oppenheimer Biotechnology, Inc. (OBI) has been aware of some unique characteristics of the Archaea for several years, and has adapted this finding into their active biodegradation consortia, the Oppenheimer Formula 1. The resulting mixture has a wide range of metabolic functions, and an increase of salt and temperature tolerance. It has been quite effective in recycling oils, sewage, grease and chlorinated compounds in water and soils in a wide range of environments.

Research has been conducted with natural bacteria cultivated on complex mixtures of hydrocarbons, and has identified a very complex nano-range microstructure for bacterial mixture activities. These mixed cultures are capable of oxidizing almost simultaneously the wide variety of the potential 300,000 natural hydrocarbon molecules that may have up to 60 carbon atoms. It is only when the hydrocarbons reach five or six rings that the microbial activity curve starts to decline.

Researchers have found it impossible to cultivate individual strains from their mixture because of the lack of knowledge of the interrelative role between each species or strain in a complex mixture, and the formulation of a medium that will allow colony development. They have tried many types of current media with little success. Textbook techniques for pure cultures or colony enumeration have failed. The organisms apparently exist in a multisymbiotic system of metabolism.

DNA and RNA sequencing techniques have been nonproductive because of the difficulty in producing pure colonies. An attempt by OBI researchers at identification, ribosomal RNA sequencing, tested the mixed culture. Only one potential match was made, but the morphological features of the Corynebacterium did not match the nanoforms of the mixed culture. Also, the organism could not be isolated, which indicated some similarity with an unidentified subcommunity in the mixture.

The microbial world is much more complex than can be anticipated by past laboratory experience in working with defined cultures because of the interreactions between microorganisms. An introduction into this unique world started with a visit to the laboratory of Prof. Perfeliev in Leningrad in 1959. His research using square capillaries introduced a new vision of the sequential evolution of microorganisms found in lake mud. His extensive pioneering work was published in B.V Perfiliev's and D.R. Gabe's 1961 text, Capillary Methods of Investigating Micro-organisms.

Illustrations in the book reveal that microorganisms, in situ, are organized in discrete groups, and not mixed, as we observe after preparing a microscope slide. It is quite obvious that each band is interfacing with adjacent bands by a form of cometabolism. Many of the forms in the book are similar to those found in ice cores. Critics of Perfeliev said that the zoning was due to Zeta potential effect, or surface potential of the bacteria and sediment. Some researchers now believe the technique revealed the true micronspace association of the Archaea and Procaryote in situ sediments. Unfortunately, Perfeliev was unable to culture most of the microorganisms observed by microscope in the square capillaries that had been stabilized at the mud water interface for 6 months or longer.

Because of the sensitivity of modern society to microorganisms, we attempt to evaluate the ecological significance of our microbial mixtures by routine screening for plant and animal pathogens. This is a default technique forced by our inadequate knowledge of this potentially vast ecosystem of unknown natural microorganisms and their associations.

The early observations in this field of microbial interaction, by Jackson Foster, established the term co-metabolism (Leadbetter, E.R. And J.W. Foster, 1960, "Oxidation products formed from gaseous alkanes by the bacterium Pseudomonas methanica", Archives for Biochemistry and Biophysics. 82:491-492). Perhaps the advanced computer with its capacity for evaluating interactions will provide the mechanism for a new technology to understand co-metabolic function. It will also require the evolution of a new breed of frontier-oriented microbiologists, willing to leave the textbook behind, who will explore the basic mysteries of this new nano-ecosystem.

Alexander, M. 1961. Introduction to Soil Microbiology. Wiley, N.Y. pp 472.

American Petroleum Institute. 1969 to present. Annual Oil Spill Conference, American Petroleum Institute, Washington, D.C.

Blank, M. Ed. 1970. "Surface Chemistry of Biological Systems." Advances in Experimental Medicine and Biology. Vol.7. Plenum Press, N.Y. pp 340.

Davis, J.B.,1967. Petroleum Microbiology., Elsevier Publishing Amsterdam, pp. 604.

Gortner, R.I. and W.A. Gortner, 1949. Outlines of Biochemistry. John Wiley and Sons, N.Y. pp 1,078.

Marr, E.K. 1959. The bacterial oxidation of benzene. Dissertation. Pennsylvania State College.

McKenna, E.J. and R.E. Kallio. 1965. "The biology of hydrocarbons." Annual Review of Microbiology., 19:183

Oppenheimer, C.H. l965. "Bacterial production of hydrocarbon-like materials." Zeitschrift fur Allgemeine Mikrobiologie, 5(4): 284-307.

Oppenheimer, C.H., Miget, R.J., and H.I. Kator., 1979. "Ecological relationships between marine microorganisms and hydrocarbons in the O.E.I. study area, Louisiana." Rice Univ. Studies, v.65, p. 287-325.

ZoBell, C.E. 1946, U.S. Patent #2,413,278. Described a process by which bacteria release oil from geological formations.

ZoBell, C.E. 1950. "Assimilation of hydrocarbons by microorganisms." Advances in Enzymology, 10:443-486.

Click here to post comments about this topic, and read what others have to say.

This article originally appeared in the November, 1999 issue of Environmental Protection magazine, Vol. 10, Number 11, pp. 34-38.

This article originally appeared in the 11/01/1999 issue of Environmental Protection.