Healthy Environments for Healthy People: by normal, selected, and/or genetically

Bioremediation Today and Tomorrow engineered microbes is emerging as a dis-

tinctive and promising approach to clean-
ing up polluted environments. Harnessing
Celia Bonaventural and Franklin M. Johnson2 microbial processes for good, rather than
1Marine Biomedical Center, Duke University Nicholas School of the experiencing their harmful attributes as
propagators of disease, is the goal of biore-
Environment Marine Laboratory, Beaufort, North Carolina; 2Division of mediation. In the following review we
Intramural Research, National Institute of Environmental Health describe current and emerging measures of
Sciences, Research Triangle Park, North Carolina remediation based on biologically active
cells and organisms and focus on microbial
Increases in environmental contamination lead to a progressive deterioration of environmental processes. Microbes play an essential role in
quality. This condition challenges our global society to find effective measures of remediation to nature's cycles and they are the primary
reverse the negative conditions that severely threaten human and environmental health. We stimulant in bioremediation of contami-
discuss the progress being made toward this goal through application of bioremediation nated environments (2-7). In the natural
techniques. Bioremediation generally utilizes microbes (bacteria, fungi, yeast, and algae), although cycles for the transformation of mercury,
higher plants are used in some applications. New bioremediation approaches are emerging based for example, bacteria are important for
on advances in molecular biology and process engineering. Bioremediation continues to be the
most of the reactions illustrated in Figure 1.
favored approach for processing biological wastes and avoiding microbial pathogenesis.
Bioremediation may also play an increasing role in concentrating metals and radioactive materials
Most elements exist in a variety of
to avoid toxicity or to recover metals for reuse. Microbes can biodegrade organic chemicals;
forms that differ in their availability and
purposeful enhancement of this natural process can aid in pollutant degradation and waste-site toxicity to humans and to other forms of
cleanup operations. Recently developed rapid-screening assays can identify organisms capable of life. To meet the challenges presented
degrading specific wastes and new gene-probe methods can ascertain their abundance at by environmental pollution, the goal of
specific sites. New tools and techniques for use of bioremediation in situ, in biofilters, and in bioremediation (together with prevention
bioreactors are contributing to the rapid growth of this field. Bioremediation has already proven and chemical and physical approaches to
itself to be a cost-effective and beneficial addition to chemical and physical methods of managing remediation) is to reduce the amount and
wastes and environmental pollutants. We anticipate that it will play an increasingly important role availability of hazardous chemical com-
as a result of new and emerging techniques and processes. Environ Health Perspect pounds and convert them to useful or at
105(Suppl 1):5-20 (1997) least innocuous products.
Key words: aerobic and anaerobic metabolism, biofiltration, bioreactors, bioremediation, Bioremediation systems generally
catabolic enzymes, in situ remediation, land farming, metal bioadsorption, pollution, selected utilize microbes (bacteria, fungi, yeast, and
and engineered microbes, sewage treatment, water quality algae) or microbial products for the degra-
dation or concentration of waste, although
in some cases higher plants are also being
developed for this purpose (8-11). Novel
Environmental Contamination the U.S. Environmental Protection Agency metabolic opportunities are introduced as a
and Bioremediation (U.S. EPA). Study results of the extra- result of species selection. For example,
and intramural research programs of the enzymes found in the fungus Phanerochaete
The publication of Rachel Carson's Silent National Institute of Environmental Health chrysosporium (white rot fungus) effectively
Spring in 1962 (1) spoke to the American Sciences have contributed to the increasing degrade some wastes that prove resistant
public about the direct link between the public awareness that human diseases often to most bacterial action (e.g., DDT and
health of the ecological environment and have preventable environmental compo- 2,4,5-trichlorophenoxyacetic acid).
the health of humanity. Since that time, all nents. Pollution prevention and environ- The need for a biological approach to
facets of American society have stepped up mental remediation are interwoven into all improve environmental conditions directly
their efforts to prevent environmental degra- strategies proposed for sustaining human relates to the increasing size of the human
dation. Congress passed the Clean Air Act and environmental health. Remediation population on a planet of finite dimen-
and the Clean Water Act and established based on pollutant metabolism or absorption sions. The estimated population of the
earth in 1996 is 6 billion people, but by
the year 2100 the number is expected to
Manuscript received 4 September 1996; manuscript accepted 10 December 1996. almost double (12). Whereas the number
Support for this work was provided by National Institute of Environmental Health Sciences Center grant
ESO-1 908. We are indebted to the Center's Editor, Shirley Tesh, for her careful reading of the manuscript. We of population doublings that might be sus-
thank Joe Wachsman and John Penta for reviewing the manuscript. The helpful comments of two anonymous tained by advances in technology (without
reviewers also resulted in improvements. We also wish to acknowledge the support provided by Sunshine bringing unbearable pain and suffering)
Makers, Inc., the manufacturers of Simple Green, for a series of workshops on environmental bioremediation may be argued, no one believes that such
that laid the groundwork for this review.
Address correspondence to Dr. C. Bonaventura, Duke University Marine Biomedical Center, 135 Duke an increase can go on indefinitely, and
Marine Lab Road, Beaufort NC 28516-9701. Telephone: (919) 504-7591. Fax: (919) 504-7648. E-mail: there are already great inequities in degree
bona@mail.duke.edu of pollution-related suffering among popu-
Abbreviations used: GE, General Electric; PCBs, polychlorinated biphenyls; RCRA, Resource Conservation
and Recovery Act; STP, sewage treatment plants; TCE, trichloroethylene; U.S. EPA, U.S. Environmental lations (13,14). As populations grow in
Protection Agency; VOCs, volatile organic compounds. size, increases in a variety of adverse human

Environmental Health Perspectives - Vol 105, Supplement * February 1997 5

 BONAVENTURA AND JOHNSON

Hg° streams, and in sediment. These pollutants treatment consists of a screening device to
are present not only in concentrated waste remove a variety of large trash and debris
sites but are widely distributed throughout (usually hauled away to landfills), a settling
the environment, although in many cases tank where coarse grit and sand particles
at levels too low to trigger regulatory action. are removed, and a primary clarifier (essen-
The kinds and amounts of these chemicals tially a large tank from which floating
are also likely to increase as populations of solids and settled sludge are removed after
humans swell. the sewage has resided in the tank for a
Bioremediation is not new to the brief period of usually a few hours). The
human race, although new approaches that limited time in the primary clarifier means
stem from advances in molecular biology that microorganisms living in the tank do
and process engineering are emerging. An not have the opportunity to consume a
important, long-standing, and increasingly large amount of the nutrient material con-
problematic bioremediation area is process- tained in the sewage. The floating solids
ing biological nitrogen waste (feces and and the sludge are then pumped to an
urine) produced by humans and the ani- anaerobic digester. The liquid effluent is
mals that humans depend on for food. As disinfected, usually with chlorine, before its
human population size, industrial produc- release into the environment. Alternatively,
tion, and chemical use have increased, so additional processes, referred to as sec-
have populations of farm animals. In North ondary- and tertiary-level sewage treat-
Carolina alone, approximately 27,000,000 ments, may be applied to further reduce the
Figure 1. The transformation, cycling, and movement tons of fresh manure containing 205,000 levels of nutrients, pathogens, and chemi-
of mercury in the environment. Mercury transforma- tons of nitrogen, 138,000 tons of phospho- cals. Of course, these additional treatments
tions are made primarily by the action of bacteria. rus, and 133,000 tons of potassium were cost money-usually from taxpayer dollars.
Physical conditions, such as temperature and pH, also generated by food animals in 1993 (18). The anaerobic digester contains micro-
have major role-determining equilibrium values in mer- Much of this waste ends up in river waters organisms adapted to grow and multiply in
cury transformations, and thus availability to man. and estuaries, where it causes enormous the absence of oxygen at elevated tempera-
Anthropogenic input of mercury into the environment problems, with secondary contributions to tures. In this process nutrients are converted
also affects the equilibrium. Unlike lead, for which there air and groundwater pollution (19-23). primarily to microbial biomass, methane,
are monitoring data to show appreciably decreased North Carolina's top health official, J.B. and carbon dioxide and thus are con-
concentrations in the atmosphere in recent years, the Howes, Secretary of the North Carolina sumed. The liberated methane is used to
concentration of mercury seems to be increasing.
State Department of Health, Environment, heat the digester. The sludge coming out of
and Natural Resources, has declared that the anaerobic digester has considerably
health and ecological effects (and associ- bad water quality, caused primarily by ani- reduced objectionable qualities (less odor
ated costs such as health care expenses) are mal and human nitrogen waste, is North as well as reduced numbers of pathogens)
also expected. Carolina's number one environmental and is typically transported to a landfill or
The U.S. EPA's Toxic Substances health problem (J.B. Howes, personal com- applied to the land as fertilizer.
Control Act Chemical Inventory includes munication). The poor water quality dead Secondary sewage treatment consists of
over 72,000 chemicals, with approximately zone in the Gulf of Mexico off the two main types: trickling filters and
2300 new chemicals submitted to the U.S. Louisiana coast reportedly covered more activated sludge. Trickling filters are cylin-
EPA every year (15,16). Along with popu- than 6000 mi2 in 1995 (24). It is no won- drical tanks containing loosely packed
lation increases, the number of different der; worldwide, the effects of poor water rocks, which range in size from 2-10 cm.
chemicals and the total amount of chemi- quality are second only to malnutrition in Effluent enters through the top; air is
cals produced are also bound to increase in the total disease burden and cause of death introduced from the bottom. Distributed
the future. In 1990, the total release of tox- of human beings (25). throughout the column is a variety of
icants into the environment by U.S. manu- Because of the importance of clean organisms that are attached to the surfaces
facturers was approximately 4.8 billion water to human health, sewage treatment of the rocks and in the intervening spaces.
pounds (17). In addition, large quantities plants (STPs) constitute the largest and Bacteria and fungi are the first to consume
of a number of toxic products are released most important bioremediation enterprise the organic constituents, and in turn the
into the environment by end users in more in the world. There are approximately bacteria and fungi are consumed by higher
or less unaltered form. These products 16,000 municipal STPs in the United trophic level organisms, including protozoa,
include those designed for household use as States, which process about 40 billion m3 rotifers, nematodes, worms, and insects.
well as industrial materials such as fuels, of raw sewage per year (5). The major Activated sludge systems consist of a series
detergents, fertilizers, dielectric fluids, components of raw sewage are suspended of tanks. Effluent is introduced at one end,
preservatives, flavorings, flame retardants, solids, organic matter, nitrogen, phospho- and it exits at the other. In between, the
heat transfer fluids, lubricants, protective rus, pathogenic microorganisms, and sewage is mixed and aerated vigorously.
coatings, propellants, pesticides, refriger- chemicals (e.g., pesticides and heavy met- Bacteria are the main decomposing organ-
ants, and many other chemicals. Such als), and even the most rudimentary STPs isms in the activated sludge system, but
materials or their breakdown products make some reductions in most of these protozoans, rotifers, and nematodes are
often accumulate in soil and aquifers near factors. A variety of methods is used for also present. All the various life forms tend
landfills and dumps, in surface lakes and sewage treatment. Generally, primary to occur together in flocculant masses.

6 Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

Both activated sludge and trickling with nitrates [NO3-, associated with with microbial pathogenesis. In England,
filter secondary STP systems can be effec- methemoglobinemia in infants, cancer, and the recent outbreak of bovine spongiform
tive, but there are advantages and disad- birth defects (36,37)] and other toxic, pos- encephalopathy (Mad Cow Disease) that is
vantages to each. Trickling filters seem to sibly carcinogenic, chemicals, including believed to be associated with Creutzfeldt-
be more tolerant of industrial chemicals, biocides (38). Other disadvantages are the Jakob disease in humans has increased con-
perhaps because of greater species/meta- increased risk of exposure to disease cern over disease transmission from food
bolic diversity. However, trickling filters pathogens and the gradual accumulation of animals to humans (47,48). In fact, a great
require more space, cost more to construct, heavy metals in soils such that the growth many microbial diseases (zoonotic dis-
and tend to create more of an odor problem. of crops can eventually become inhibited eases) can and often do cross over to affect
Activated sludge systems tend to achieve (39). In spite of these problems, land humans. Diseases that can pass to humans
greater reductions in organic nutrients and application of STP effluent has been from swine, for example, include:
suspended solids. remarkably useful in many cases, e.g., the * bacterial infections, such as anthrax
Regardless of which secondary process reclamation of strip-mined soil (40,41). (Bacillus antracis), brucellosis (Brucellosis
is used, without further (i.e., tertiary) treat- It is estimated that more than half of suis), ampylobacteriosis (Campsylobacter
ment, large amounts of nitrogen and phos- the rainwater that falls on the United jejuni), erysipeloid (Erysipelothrix
phorus remain in secondary STP effluent States is converted to wastewater by peo- rhusiopathiae)
(26). These inorganic nutrients in turn ple, cities, and industry (42). Although * viral infections, such as encephalomy-
encourage algal and phytoplanktonic there are many less-than-ideal systems, ocarditis (Cardiovirus), influenza
growth in receiving waters. Ultimately, bioremediation carried out in STPs does a (Influenzavirus), Japanese B encephali-
these organisms die and decompose, which reasonable overall job of cleaning up this tis [Flavivirus (gp A)], and vesicular
consumes oxygen and thereby promotes huge amount of waste. Agricultural opera- stomatitis ( Vesiculovirus)
hypoxic and anoxic conditions. Fish kills tions, on the other hand, sometimes do not * nematode infections, such as ascar-
resulting from oxygen deprivation are tend to their animal wastes. Sixty percent iasis (Ascaris suum) and trichinosis
notable consequences; in extreme cases, of water quality impairment is attributed (Trichinella spp.)
millions of fish are killed (23,27,28). The to silt and fertilizer runoff (42). * protozoan infections, such as balantidi-
technology to remove both nitrogen and Septic tanks are another large and asis (Balantidium coli), toxoplasmosis
phosphorus (and as a result, counteract imperfect bioremedial system that con- (Toxoplasma gondii), amoebic dysen-
these effects) has been available for some tributes nitrogen and other waste to the tary/amebiasis (Entamoeba polecki) and
time (29). Inorganic phosphorus can be impairment of water quality, particularly to sarcocystosis (Sarcocystis suihominis)
precipitated from solution by the addition groundwater. Patrick et al. (43) estimated * spirochetal infections, such as leptospi-
of calcium (as lime, CaO), aluminum (as that almost one-third of the U.S. popula- rosis (Leptospira interrogans) (49,50).
alum, aluminum sulfate), or a variety of tion still relies on septic tanks and that they Although the advent and continued
other relatively inexpensive chemicals. handle roughly 15 billion liters per day of development of antibiotics have kept infec-
Nitrogen can be removed both chemically sewage. U.S. EPA studies (44) indicate tious disease in developed countries under
and biologically. Most of the nitrogen in that about one-third of all septic tanks control for many years, there is growing
secondary sewage effluent occurs as ammo- operate improperly; as a result septic tanks evidence that this may not be effective
nium ion (NH4+). The process of ammo- are the primary source of groundwater con- indefinitely, as increasingly virulent and
nia stripping involves the conversion of tamination in many parts of the country. antibiotic-resistant strains continue to
NH4+ to ammonia gas (NH3) by raising This contamination leads to nitrate, chem- evolve (51-53).
the pH along with vigorous agitation. icals, and pathogens in the well water that Two additional classes of pollutants
However, the liberated ammonia gas then some people drink. also continue to be of enormous practical
becomes a potential atmospheric pollu- The risk of pathogenic disease associ- and economic importance and as a result
tant. Biological conversion of nitrogen gas ated with nitrogen waste should not be merit special mention in any discussion of
(N2) by denitrifying bacteria is an alterna- underestimated. Human waste, in the form bioremediation. The first is the inorganic
tive approach, although there are other of sewage treatment plant sludge, for pollutant category of heavy metals, such as
approaches as well, e.g., break point chlori- instance, contains significant amounts of lead, mercury, and cadmium. These nat-
nation, reverse osmosis, and distillation bacterial, viral, and other pathogens even ural elements, found in the Earth's crust,
(30,31). In spite of the available technol- after stabilization and treatment (45). are utilized in many industrial processes
ogy, implementation has been limited, and Some of these pathogens can remain viable and products, a use which has resulted in
eutrophication, caused in part by the efflu- for long periods of time and contaminate their release in higher concentrations and
ent of STPs, still commonly occurs in many groundwater located below the sludge in more accessible form than is typical in
coastal regions throughout the world. deposit locations. Huge areas deep beneath natural systems. Incorporation of heavy
The discharge of STP effluent on land the sea still contain viable pathogens as a metals into inorganic and organometallic
rather than in water has been tried many result of offshore ocean disposal of sewage complexes often alters their biological
times, often with at least initial success waste (46). Pathogens are also found in activity; such changes are just as likely to
(32-35). The potential advantages of land animal waste, and the presence of nitrogen increase toxicity, due to increased bioavail-
deposition are that groundwater resources together with microbial contamination ability, as they are to decrease toxicity.
can be recharged and that valuable nutri- may exacerbate the expression of disease. Furthermore, depending on conditions of
ents become available to assist with crop Improved bioremediation of biological pH, increased temperature, etc., natural
growth and other vegetation. Disadvantages wastes is envisioned as a necessary first step cycles may intervene to convert or mobilize
include possible groundwater contamination in breaking the chain of events associated relatively benign inorganic species to more

Environmental Health Perspectives - Vol 105, Supplement 1 * February 1997 7

 BONAVENTURA AND JOHNSON

toxic organic complexes, e.g., conversion of ultimately yield other radioactive metabo- removing lead from gasoline. For other
elemental mercury to methylmercury lites, which can be as hazardous or more so heavy metals, the results are less encourag-
(Figure 1). than the parent compound. However, cer- ing. Mercury, for instance, seems to be
A second class of pollutants is radioac- tain radioactive substances, such as ura- increasing in the environment in spite of
tive waste materials. Radioactive com- nium, can be immobilized, concentrated, environmental control measures (60-62).
pounds, although they may be biologically and removed from the environment with There has been great interest in
converted from one form to another, the aid of suitably adapted microorganisms, mercury contamination since the 1950s,
e.g., Citrobacter sp. (54) or appropriate when hundreds of people in Japan became
0.90-s biomass (55-58). seriously ill and many died from eating
0.80 -
Unlike organic pollutants, the toxicity seafood that was contaminated with
of metals is inherent in their atomic struc- methylmercury. North Carolina is one of
0.70 -
ture, and they cannot be further trans- many states in the United States that now
c, 0.60- muted/mineralized to a totally innocuous routinely monitor freshwater fish for mer-
a, 0.50- form. Their oxidation state, solubility, cury levels. Many fresh- and saltwater fish
0L
0.40- and association with other inorganic and have mercury levels greater than the alert
0.30- organic molecules can vary, however; levels designated by the U.S. Food and
0.20- microbes as well as higher organisms may Drug Administration and the World Health
0.10- play a bioremediative role by concentrating Organization. Fish in North Carolina with
metals so that they are less available and reported mercury concentrations greater
72 74 76 78 80 82 84 86 less dangerous. than 1 ppm (range: 1-6.9 ppm) in the
Years Because of the proven health danger of edible (filet) portion include the following:
heavy metals, federal and state governments * Ictaluruspunctatus (channel catfish)
Figure 2. Average yearly lead (Pb) concentrations in routinely monitor the environment for * Ictalurus (Ameriurus) catus (white catfish)
ambient air in North Carolina. The decline represents their presence. In cases where control strate- * Ictalurus (Ameriurus) natalis (yellow
the results of removing lead from gasoline. Currently, gies have been implemented, there have bullhead)
the most important sources of lead in air are thought to been significant decreases in environmental * Ictalurus (Ameriurus) nebulosus (brown
result from the sandblasting of bridges and water
tanks, many of which are still coated with lead-based metal levels. Figure 2 shows recent levels of bullhead)
paint. Children playing in lead contaminated areas are atmospheric lead in North Carolina (59). * Ictalurus (Ameriurus) platycephalus (flat
particularly at risk. The sharp decline is largely the result of bullhead)

I
N

/00,10"

River basins
Broad New
Cape Fear Pasquotank
Catawba Roanoke [Hglppm

Chowan Savannah | [Hg]>1 ppm

French Broad Tar-Pamlico
Hiwassee Watauga
LitdeTennessee White Oak
Lumber Yadkin-Pee Dee
z Neuse Albemarle Sound-local tributaries
Figure 3. Map of North Carolina showing sampling sites from which fish were collected and analyzed for the presence of mercury. The state's watersheds are indicated by
color. Sites where some fish contained greater than 1 ppm mercury are indicated, as are sites where some fish contained less than 1 ppm mercury.

8 Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

* Lepisosteus osseus (longnose gar) and Baker and Brooks (71). As these and considerable amounts of Hg0 relative to
* Carassius auratus (goldfish) other authors, e.g., Shann (72), point out, control plants. However, the transforma-
* Micropterus salmoides (largemouth bass) such organisms may provide the opportu- tion of ionic mercury to the metallic ele-
* Cyprinus carpio (common carp) nity to return waste material to useful prod- mental form, which then volatilizes to
* Pomoxis nigromaculatus (black crappie) ucts rather than merely transform them to become an air pollutant, is a less than ideal
* Lepomis gulosus (warmouth) innocuous substances. However, a practical remedial solution.
* Lepomis auritus (redbreast sunfish) phytoremedial technology remains to be The recovery of metals by microbes or
* Lepomis gibbosus (pumpkinseed sunfish) developed, although progress has been microbial products is more advanced. For
* Lepomis macrochirus (bluegill) made with transgenic Arabidopsis thaliana example, many microorganisms secrete
* Esox niger (chain pickerel) expressing merApe9 (73). Grown on high-affinity, metal-binding compounds
* Amia calva (bowfin) medium containing HgCl2, at concentra- called siderophores. The siderophores bind
* Carcharhinus obscurus (dusky shark) tions of 25 to 100 M (5-20 ppm), these specific chemical forms of metals, and the
* Carcharhinusplumbeus (sandbar shark) transgenic merApe9 seedlings evolved metal-siderophore complex then absorbs
* Carcharhinus limbatus (black-tipped
shark)
* Odentaspis taurus (sandtiger shark) Table 1. Examples of plants that hyperaccumulate metal.
* Sphyrna lewini (scalloped hammerhead Genus and species Family Metal concentration, pg/ga.b
shark) Cobalt
* Galeocerdo curvieri (tiger shark) Aeollanthus biformifolius Lamiaceae 2820
* Makaira nigricans (blue marlin) Alectra sessiliflora Scrophulariaceae 2782
Figure 3 is a map of North Carolina Anisopappus davyi Asteraceae 2650
showing sampling sites where fish with Crotalaria cobalticola Fabaceae 3010
1 ppm or more mercury have been found. Cyanotis longifolia Commelinaceae 4200
These data were collected by the North Haumaniastrum homblei Lamiaceae 2633
Haumaniastrum robertii Lamiaceae 10200
Carolina State Department of Health,
Environment and Natural Resources, Copper
Aeollanthus biformifolius Lamiaceae 3920
beginning in 1980. They are available Buchnera henriquesii Scrophulariceae 3520
under the STORET database (U.S. EPA Bulbostylis mucronata Cyperaceae 7783
Water Data Storage and Retrieval Data Eragrostis boehmii Poaceae 2800
Base, U.S. EPA, Research Triangle Park, Gutenbergia cupricola Asteraceae 5095
NC) maintained by the U.S. EPA but are Haumaniastrum katangense Lamiaceae 8356
otherwise previously unpublished. The lpomoea alpina Convolvulaceae 12300
Lindemia perennis Scrophulariaceae 9322
data show that there are no locations in Pandiaka metallorum Amaranthaceae 6260
North Carolina that can be considered free Vigna dolomitica Fabaceae 3000
of a potential mercury problem. Although Nickel
no adverse human effects attributable to Alyssum argenteum Brassicaceae 29400
mercury poisoning have been reported Allyssum masmenaeum Brassicaceae 24300
in North Carolina, some areas have Bommueller baldacci tymphaea Brassicaceae 31200
posted signs to warn people not to con- Geissois intermedia Cunoniaceae 22900
sume large amounts of fish. This problem Geissois pruinosa Cunoniaceae 34000
Hybanthus austrocaledonicus Violaceae 25500
occurs in other parts of the country, Peltaria emarginata Brassicaceae 34400
e.g., Arkansas, South Carolina, Georgia, Phyllanthus serpentinus Euphorbiaceae 38100
Florida, Wisconsin, and the Great Lakes Psychotria douarrei Rubiaceae 47500
region (63-68). Declines in populations of Thlaspi alpinum sylvium Brassicaceae 31000
fish-eating wildlife, such as loons, mink, Lead
otter, and wild cats are possibly attribut- Armeria maritima halleri Plumbaginaceae 1600
able, in part, to environmental chemicals Thlaspi alpestre Brassicaceae 2740
such as mercury. In humans the subtle Thlaspi rotundifolium cepaeifolium Brassicaceae 8200
neurological effects of chronic low-level Manganese
mercury poisoning might not be obvious. Macadamia neurophylla Proteacea 51800
Maytenus bureauvianus Celastraceae 33800
Many plants and bacteria have evolved Maytenus sebertiana Celastraceae 22500
various means of extracting essential nutri- Zinc
ents, including metals, from their environ- Thlaspi alpestre Brassicaceae 25000
ment. In the course of prospecting for Thlaspi calaminare Brassicaceae 39600
minerals, unusually tolerant species have Thlaspi caerulescens Brassicaceae 27300
been observed in the vicinity of metal-rich Thlaspi tatraense Brassicaceae 27000
deposits. In some cases, these tolerant Mercury
organisms concentrate metals several thou- Jungermannia vulcanicola (Liverworts) 13000C
sandfold over ambient concentrations. Scapania undulata (Liverworts) 4700d
Some examples are listed in Table 1 and in "Dry weight basis. bData from Baker aand Brooks (71) except where noted. cData from Satake and Miyasaka (139).
Zajic (69), Antonovics and Bradshaw (70), dData from Sameck-Cymerman and Keempers (140).

Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997 9

 BONAVENTURA AND JOHNSON

back into the organism for xutilization. such as arsenic, cadmium, chromium, biodegradation pathways of a number of
Siderophores are an evolution ary adapta- cobalt, copper, nickel, mercury, tellurium, pollutant classes; recent reviews have been
tion used particularly for suppl'ying iron to and zinc (79). Appanna et al. (80) recently published for many, including polycyclic
cells (74). Iron is an essential e lement that described a Pseudomonas fluorescens model aromatic hydrocarbons (85), polychlori-
is often not bioavailable in sufficient with potential application in the manage- nated biphenyls (PCBs) (86,87), and pes-
amounts in the environment. AL bioremedi- ment of cesium from nuclear industries. ticides (88,89). Rapid screening assays are
ation technology in developme nt will deal Practicable bioremediation technologies being developed by researchers to identify
with metal pollution and miake use of exploiting these abilities have yet to be per- organisms capable of degrading specific
native and chemically modifFied sidero- fected, but the potential for metal-concen- wastes (90). Molecular probes make it pos-
phores attached to inert supF?ort media. trating bioreactors has been recognized and sible to test a small, mixed microbial popu-
This approach has been used with some is the object of current research. lation for specific degradative enzyme genes
success in the removal of plut( Dnium, ura- Perhaps the best-known example of (91). Gene probing can also give an indica-
nium, and thorium from radio active waste microbial metal metabolism is the mining tion of the natural abundance of organisms
(75). Premuzic et al. (76) disc-overed that microbe Thiobacillus ferrooxidans (81,82). with the potential to degrade specific
uranium and thorium induce siderophore This bacterium and other related species pollutants at a given site.
production in Pseudomonas aeroginosa. derive energy by breaking down metallic Some organisms have the capacity to
Enterochelin (Figure 4) is an example of a sulfides. Many heavy-metal ores, such as utilize an environmental contaminant as a
siderophore (77). copper, gold, cadmium, lead, mercury, food source and thus grow and multiply
Another bioremediative meXchanism to uranium, and zinc, often occur as com- more prolifically in areas where the con-
deal with metal contaminatio n is bioad- plexes with sulfur. Microbial metabolism taminant is present. Mutation and selec-
sorption. Many bacteria and aLlgae have a of the sulfide releases the free metal; sulfu- tion may also result in the evolution of
cell wall or envelope that is capable of ric acid is produced as a by-product, which microbial strains adapted to utilize envi-
passively adsorbing rather hitgh levels of contributes to further metal leaching. ronmental contaminants. Consequently,
dissolved metals, usually via a c]harge-medi- Technologies utilizing T. ferrooxidans as organisms with the highly developed capa-
ated attraction. Cell-wall const:ituents that an inexpensive, nonpolluting alternative to bility to metabolize specific contaminants
make this possible include ca.rbohydrate high-temperature ore smelting have been can be found in contaminated sites. These
polymers, peptidoglycans, an d melanins in limited use since the late 1970s, and organisms can be isolated from the micro-
(78). Other bacteria absorb and utilize development of larger scale applications biological population and cultured for
mineral complexes and have evc)lved special continues (83). Proposed bioremedial uses inoculation of other sites or for use in
ways of packaging and handlinig the resid- of sulfur-oxidizing bacteria include bioreactors. This approach has successfully
ual metabolites containing heavry metal as a removal of sulfur from coal and fuel oil isolated bacterial strains capable of metabo-
protection against metal toxi city. Often and use in biofilters to scrub sulfur dioxide lizing marine crude-oil spills (92), jet fuel
this process culminates in s ecretion of (SO2) from various industrial emissions. (93), and organohalogens in groundwater
heavy-metal precipitates or ins(oluble com- Although heavy metals are not actually aquifers (94,95). Organisms isolated in
plexes. Manipulation of nutrieent levels in accumulated by such bacteria, there is this way have been used in a number of
culture and environmental coriditions can potential for bioreactor treatment of land treatment and bioreactor systems.
stimulate such activity and lead1 to remark- metal-contaminated solid wastes, thus In contrast to the selection of geneti-
able concentration efficiencie<s for metals resulting in extraction of valuable metals cally able microorganisms found in a nat-
for reuse, especially when sulfides are also ural environment, a number of researchers
present in high concentration. Bioreactor focus on developing genetically engineered
0
designs that utilize sulfide-oxidizing strains of microorganisms where deliberate
microbes to release heavy metals from con- manipulation of DNA sequences yields
0_-C-CH2 NH
~ 0 taminated soils and sludges are now being capabilities that the organisms did not pre-
c explored, with promising early results viously possess. Although genetically engi-
0
(84). Other bacteria catalyze the reverse neered microbes hold considerable promise,
reaction: anaerobic reduction of sulfates to their use in bioremediation applications
C
-H sulfides. Sulfides produced in this manner will require further study to clarify issues of
2 readily complex with heavy metals and safety and containment (96).
precipitate. Many commercial ore deposits
are believed to have been produced by bio- Bioremediation Now?
genesis (69). Sulfate-reducing bacteria Environmental contamination results in
I C NH may prove to be valuable for precipitation
of dissolved metals in waste slurries in
increased health-care costs because many
human health problems have an environ-
HC anaerobic bioreactor technology. mental component and, according to some
CH 2'--~ _-~ CH experts, the health-care system is already
_
'I Adaptable Microbial failing and in crisis (97). In addition, the
Metabolism loss of biodiversity resulting from degrada-
Figure 4. Enterochelin, shown binding an atom of iron, Microbes can be encouraged to biodegrade tion of environmental health threatens
is a siderophore produced by Klebsiella pneumoniae. almost any organic chemical. Environmental multibillion dollar global industries, such
Microorganisms secrete and reabsorb siderophores as chemists and microbial ecologists have as agriculture, biotechnology, pharma-
means of obtaining iron. extensively characterized the natural ceuticals, and nature tourism (98). Recent

I10 Environmental Health Perspectives * Vol 105, Supplement I * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

emphasis on sustainable development has comparisons. Since bioremediation can General Considerations
focused global attention on the need to ideally destroy organic wastes without Bioremediation applies appropriate
adopt environmentally friendly industrial creating adverse residues, such considera- microbes or higher organisms to undesired
approaches, not only to maintain the tions may favor bioremediation over contaminants and manipulates conditions
Earth's life-support systems, but to ensure nonbiological alternatives. to maximize the desired activity. Non-
the future of natural resources and the biological methods have been effectively
economies that they support (98). Bioremediation combined with biological methods to
It is proving to be not only environ- and Biodegradation enhance degradation of recalcitrant pollu-
mentally advantageous but also politi- Environmental bioremediation, as noted tants. For example, chemical peroxidation
cally and economically sound to clean up above, refers to a process of environmental of organohalogens has been demonstrated to
polluted air and water systems. Enact- improvement in which biological organ- increase bacterial degradation dramatically
ment of federal legislation, such as the isms or products play a key role (2-4). (99). No single method can be expected
Comprehensive Environmental Response, Many microorganisms can adapt their to work equally well on all constituents of
Compensation, and Liability Act of 1980 catabolic machinery to make use of unde- a diverse mixture; a combination of reme-
and the Superfund Amendments and sirable environmental pollutants as food diation approaches may represent the most
Reauthorization Act of 1986, has made sources, thereby degrading the environ- cost-effective solution among several
responsible parties liable for the costs of mental pollutant from an energy-rich state practicable ones.
waste-site cleanup-costs that can be enor- to an energy-poor one. Thus, microbes The methods most appropriate for con-
mous. In addition to actual Superfund bioremediate the environment as they centration and containment of pollutants
remediation, efforts to avoid liability biodegrade the pollutant to obtain energy. depend greatly on water content. Traditional
for EPA-administered cleanups under The terms bioremediation and biodegra- approaches have involved incineration and
the terms of these statutes have led to large- dation are sometimes erroneously treated wet oxidation with controlled emission in
scale remediation efforts by private ind- as synonymous. Biodegradation specifi- order to capture the contaminants. When
ustrial concerns in the United States. cally refers to chemical breakdown or min- the water content is high, these methods
Therefore, an obvious motivation to reme- eralization of materials (not necessarily do not work well and contaminant concen-
diate is legal compliance. The immediate waste) facilitated by biological organisms tration by bioaccumulation may be an
stimulus may be direct regulatory agency or products. The results of this action may attractive alternative. In situ bioaccumula-
action or cleanup requirements related to or may not be judged to be remediative, tion by certain plants is a promising tech-
real estate transactions. In many states although biodegradation of waste often nology for dealing with slowly leaching
property titles cannot be legally transferred leads to bioremediation. Biodegradation of metal sources such as mine drainage. The
unless the liability for any contamination organic chemicals occurs enzymatically. bioaccumulating organisms become loaded
discovered and cleanup required is assigned. Catabolic enzymes are proteins that cat- with toxic substances, and the resulting
The increased need for remediation of alyze specific degradation reactions of biomass, whether it is bacterial or plant,
diverse classes of waste and waste sites has organic molecules and usually display high must be collected and disposed of before it
created a demand for improved remedia- affinity for certain substrates. By this decays. Bioremediation of radioactive pol-
tion techniques and for techniques that are process, organic wastes (and some inorganic lutants is a developing field of great impor-
applicable to a wider variety of cases. As an wastes) may be absorbed by the microbe tance for the future acceptance of nuclear
emerging technology, bioremediation is and internally broken down, or wastes may power. Reviews of the interactions between
poised for rapid development. be degraded externally by secreted enzymes, radioactive substances and microorganisms
A second motivation for bioremedia- after which the metabolites are absorbed and microbial products have been pub-
tion is the dramatic increase in the cost of and utilized. Alternatively, microbial cata- lished (77,100,101). Radioactive metals
traditional waste treatment methods, espe- bolic enzymes, produced for the break- may potentially be concentrated and
cially bulk disposal by incineration or down of normal food sources, may degrade reprocessed in the same ways as ordinary
landfill. Bioremediation is, in many situa- certain wastes that are present as well. This heavy metals.
tions, a more cost-effective approach than process, termed co-metabolism, requires an
containment or treatment by traditional ample supply of the preferred food sub- Categories of
chemical, physical, or thermal processes. strate and also requires that the degrading Bioremediation Technology
For complex mixtures of waste, biode- enzymes come into contact with the waste Three categories of bioremediation tech-
gradative treatment offers a typical savings by waste absorption or by enzyme secre- niques have been identified. They involve
of 60 to 90% over landfill disposal costs. tion. Either direct metabolism or co- distinct technologies for remediation in
Accurate financial comparisons between metabolism can be enhanced in rate and situ and for use of biofilters and bioreac-
waste-treatment techniques require analy- extent over that which occurs naturally by tors. The first category, in situ land treat-
sis of specific processes and specific waste measures that boost the microbial popula- ment-treatment of contaminated material
compositions. Whether the method is tion or increase the availability of food in place-is a method for bioremediation
biological or nonbiological, the possibility and/or growth-limiting nutrients. The of contaminated soil and, to some extent,
of incomplete remediation exists. Toxic basic information required to enhance nat- of associated groundwater. The action may
by-products or residues of primary treat- ural biodegradative processes is knowledge be as simple as nutrient enrichment or may
ment (incinerator fly ash, chemical sludge, of the microorganisms present in a given involve further manipulation of site condi-
spent filters and scrubbers, etc.) may site, their growth requirements, and how tions, such as inoculation of the contami-
themselves be subject to a final disposal these organisms interact with one another nated site with selected microorganisms,
step. Such costs must be included in and their environment. mixing and aeration of surface soils, or pH

Environmental Health Perspectives * Vol 105, Supplement * February 1997 II

 BONAVENTURA AND JOHNSON

modification. In situ treatment involves a minimal. When accumulating waste levels Additionally, variations in reactor condi-
minimum amount of effort and cost since become toxic to the microbes or when tions, e.g., oxygen concentration, tempera-
the contaminated material does not have to nutrients from the organic medium are ture, and pH, can affect not only the rate of
be contained or extensively handled. The depleted, the filter bed is changed. This is degradation, but also the identity of the
expensive and potentially dangerous job of the only biological technique currently metabolites produced. In contrast to land
transporting waste is eliminated, and fur- available to remediate airborne pollutants. treatment, bioreactors require better knowl-
ther disturbance of the site itself is mini- Application of biofilter technology is at edge of both the exact composition of the
mized. The chief drawback to this approach present limited to removing volatile waste to be degraded and the degradation
is that degradation monitoring is difficult, organic compounds (VOCs) from point pathways catalyzed by the microbes in use.
and concerns about toxicant dispersal may sources only. Waste remediation technolo- Portable bioreactor units permit a type of
rule out such methods altogether. A related gies for treatment of ambient air pollution ex situ bioreactor treatment with minimal
bioremediation approach, referred to as are essentially nonexistent. transportation costs. This technique has per-
land farming, is applicable to solids, Bioreactors are the third and most tech- haps the greatest potential for use as a rou-
sludges, liquids, or contaminated soil that nologically sophisticated category of envi- tine treatment method for industrial wastes.
is treated at a designated site. The wastes ronmental bioremediation. Bioreactors Waste effluent streams can be treated at the
are sprayed onto, plowed into, or otherwise offer a much faster means of waste biode- site of production by directing them into a
mixed with surface soils; aeration, water, gradation than land treatment and more bioreactor process line.
and nutrient enrichment are applied to control over reaction conditions and efflu- In aqueous solution, dispersants that
achieve the best conditions for microbial ent quality than simple biofilters. In con- increase the bioavailability of organic
and/or plant growth. Land farming is iden- trast to the months or years required for wastes to microbes may also be employed.
tical in practice to in situ treatment of con- land treatment, bioreactors may require In many cases the degradation process pro-
taminated soil, except that treatment takes only days or weeks for effective degradation ceeds rapidly at first, then slows over time.
place at a different location from the site of of specific pollutants. Slurry-phase bioreac- This rate change may be explained by
contamination. Waste transport allows the tors are suitable for remediation of high product inhibition of microbial action or
use of a dedicated remediation facility concentrations of soluble organic wastes in by the presence of waste components that
where various considerations, such as con- soil or sludges; these reactors are recog- are tightly bound on or within soil parti-
tainment, monitoring, accessibility, secu- nized as capable of handling up to at least cles. Surfactants and other chemicals that
rity, etc., can be optimized. In most cases, 250 g/kg levels of organic wastes (103). accelerate desorption can be used in biore-
however, land farming as a final treatment Recalcitrant wastes, such as organohalo- actor systems to increase the degradation of
for wastes is banned in the United States by gens, may require pretreatment; some tightly bound materials. For more in-depth
the terms of the 1984 Hazardous and Solid wastes, such as organic corrosives and most treatment and discussion of other consider-
Waste Amendments and the 1976 Resource inorganics, may not be degradable by ations of reactor dynamics see Weber and
Conservation and Recovery Act (RCRA). current bioreactor systems. DiGiano (7).
The so-called RCRA land ban prohibits any There are many varied bioreactor
form of waste disposal on land unless the designs, which allows for treatment of a Applications of
waste has been treated to prescribed stan- wide variety of wastes with varied water Bioremediation Methods
dards. Thus, land farming can currently be and organic content. In some designs bac- Enhanced soil bioremediation may be
used only as an intermediate treatment step terial growth is optimized in a well-mixed achieved as simply as adjusting nutrient,
to be followed by further treatment or dis- aqueous phase contained in a lagoon, tank, pH, water, and oxygen levels to encourage
posal (102). As with in situ land treatment, or other reactor vessel into which slurries growth of indigenous soil microbes by fer-
land farming requires large amounts of ded- of waste or contaminated material are tilizing, irrigating, and tilling the soil.
icated land and usually months or years introduced. Other bioreactor designs Tilling to achieve greater mixing between
before bioremediation is complete. specifically limit mixing. Control over contaminants and microbes can become
The second category of bioremediation mixing, aeration, temperature, nutrient an elaborate process if large areas are
consists of biofiltration methods for treat- levels, water content, etc., is increased dra- involved. In some cases, forced aeration of
ing gaseous streams. The usual biofilter matically over that which is possible with surface soils with compressors or vacuum
design is a permeable organic filter bed, land treatment techniques. Degradation pumps may be applied. Extensive charac-
which serves as a culture medium for monitoring is made easier because the sys- terization of soil and waste parameters can
microorganisms. Nonliving immobilized tem is contained and output is regulated. be used to determine exactly what enhance-
enzyme biofilters are also possible biofilters. Furthermore, control over release of non- ment measures are necessary or if bioreme-
Biofilters may concentrate pollutants in a indigenous organisms to the environment diation is feasible (104,105). Inoculation
manner analogous to conventional stack is possible. The bioreactor approach is of soil with microbial strains selected for
gas scrubbers or may be used in some cases more expensive than in situ or land treat- their particular abilities may appear to offer
to biodegrade contaminants to a nontoxic ment methods because specialized reactors the greatest potential for desired biodegrada-
form that is compatible with natural envi- and equipment are utilized and materials tive action. In many cases, such techniques
ronmental processes. The latter holds the must be more extensively handled and prove ineffective due to incompatibilities
advantage of actual reduction of waste sorted. Bioreactor operation requires much between microbes and environmental con-
rather than simply repackaging for later attention and expertise to achieve the rapid ditions or due to technical limitations on
disposal. Such a biofilter can be thought of degradation that offsets the higher costs inoculant size (106,107). Unwanted sur-
as a type of biological catalytic converter. (7). Maintenance of optimum conditions prises may also occur, such as growth of a
As a general rule, operator intervention is for microbial activity is far from trivial. variant opportunistic pathogen that grows

12 Environmental Health Perspectives * Vol 105, Supplement * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

particularly well on a common organic that pump-and-treat measures can be effec- water can be nutrient- and oxygen-
contaminant, e.g., gasoline (108). tive has led to an appreciable effort in this enriched prior to recharge, stimulating
Bioreactors for remediation of contami- direction at numerous hazardous waste aerobic biodegradation of soil-bound,
nated soil offer advantages in enhancing the sites. However, the goal of remediating water-insoluble wastes by indigenous soil
transport of both nutrients and waste to the aquifers to drinking water standards by microorganisms. Actual oxygen content of
degrading microorganisms. Furthermore, such techniques may be unrealistic in the water can be boosted by air pumps, or
bioreactors can be completely contained many, if not most, cases. Curtis C. Travis, alternative oxygen sources such as hydro-
and all outputs regulated, induding gaseous director of the Center for Risk Management gen peroxide may be added. Surfactants
emission of volatile wastes and metabolites. and head of the Risk Analysis Section of and other organic waste desorbing chemi-
Soil slurries can be oxygen saturated for aer- the Health and Safety Research Division at cals can also be added to increase waste
obic degradation or anoxic for anaerobic Oak Ridge National Laboratory, Oak bioavailability. If a surface bioreactor is
degradation, as desired. Organisms can Ridge, Tennessee, recently addressed the used, some portion of the active microbial
thus be utilized in bioreactors that would question of whether it is technically possi- biomass can be recharged with the water,
never thrive or even -survive in some soil ble to restore contaminated groundwater providing continuous inoculation of the
environments. Because of the containment to an environmentally sound condition contaminated aquifer and soil. Although
this method affords, it is the most likely (110). He strongly argues that pump-and- stimulation of aerobic metabolism is the
bioremediation procedure to make use of treat technologies cannot do the job. objective of most systems, the reinjected
genetically engineered microbes. Contamination levels at remediation sites groundwater can be enriched with nitrate
Selected or genetically engineered are typically two to three orders of magni- to stimulate growth and enhance the
microbes that produce the high-affinity tude above allowable drinking water limits. biodegradative action of anaerobic denitri-
metal-binding protein metallothionein Based on practical experience gained in fying microbes. Recently, this approach has
have been suggested as a biological metal- pumping and treating contaminated proved effective in degrading the various
sequestering system for waste water treat- aquifers over the past 10 years, Travis organic constituents of gasoline, with toxi-
ment (109). Production of metal-binding argues that treatment typically drops pollu- city reductions comparable to those seen in
proteins by cultured microorganisms for tant concentrations by a factor of 2 to 10, aerobic degradation (111). As with in situ
subsequent extraction is also potentially then levels out with no further decline. soil treatment, the success of subsurface
useful for treatment of metal-contaminated Cessation of pumping is often followed by aquifer bioremediation is largely deter-
wastes and environments. Immobilized a rebound in aquifer waste concentrations. mined by waste and soil characteristics.
metallothioneins could be used in biofilters The problem is largely that sites are typi- Soil permeability is especially important to
and bioreactors to remove dissolved metals cally contaminated with organic wastes the success of nutrient enrichment and
from waste streams. that do not readily dissolve in the aqueous inoculation efforts.
Contaminated subsurface aquifers phase. The waste either remains adsorbed Over the years, a number of lakes and
frequently accompany soil contamination. to the soil matrix, floats on the top, or rivers have become seriously contaminated
Bioremediation of groundwater resources sinks to the bottom of the water table. with various industrial wastes in many
presents unique problems and risks. Among Therefore, wastes only slowly seep into the parts of the United States. In some cases,
the most obvious of these problems are groundwater at a diffusion-limited rate, the sources of pollution have been reduced
that groundwater is mobile, whereas soil and cannot be significantly changed by or even eliminated. Public demand for
is generally stationary, and that people groundwater pumping. Pump-and-treat remediation to a condition safe for fishing
and livestock frequently drink untreated measures may dramatically reduce pollu- and other recreational uses is growing.
groundwater. Thus, there will often be an tant concentration in the aqueous phase of Unfortunately, technologies for surface
additional urgency factor associated with the aquifer; when the pumps are switched water remediation are not nearly as well
groundwater cleanup that may justify more off, however, pollutants gradually leach out developed as those for soil or even ground-
drastic and expensive measures. of the soil and the aqueous concentration water. Part of the problem lies in the size of
The usual approach in remediation of rises again. Many leading hydrologists have many bodies of water. It is technically and
contaminated aquifers is groundwater concluded that hundreds to thousands of environmentally impractical to divert a
pumping and surface treatment to elimi- years of pumping could be required to large flowing body of water from its course
nate the water-soluble wastes. The treated purge some contaminated aquifers of their for treatment. Also, as with underground
water is then recharged into the aquifer via organic waste contaminants (CW Hall, aquifers, the water contamination problem
one or more injection wells at some point unpublished data). Travis draws the unfor- is largely a sediment contamination prob-
upgradient to the contaminated zone. tunate conclusion that "Not a single lem. Many persistent wastes become tightly
Pump-and-treat operations can incorporate aquifer in the United States has been con- bound to bottom sediments from which
bioremediation in at least two ways. The firmed to be successfully restored through they slowly leach out, and thus cannot
most obvious method uses biological pumping and treating" (110). The impli- readily be removed by water treatment.
(bioreactor) surface treatment; but, like any cation is that, although pump-and-treat Conventional treatment typically involves
pump-and-treat approach, this method is measures may be useul to limit dispersal of dredging and removing bottom sediment
only able to degrade wastes in the mobile, a waste plume into the water table, massive in the most polluted areas, but such mea-
aqueous phase. It is important to recognize excavation of soil is usually required to sures can themselves be environmentally
that many organic wastes have low water remove the source of the problem. devastating and there is a risk of remobiliz-
solubilities, and aquifer-associated soils will A more recently developed bioreme- ing toxicants accumulated over many years.
often contain larger volumes of organic diation approach to water treatment is sub- Furthermore, the excavated sediment must
wastes than the water itself. The conviction surface in situ remediation. The treated still be treated and/or disposed of as toxic

Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997 13

 BONAVENTURA AND JOHNSON

waste. Workers are turning to in situ related to remediation of marine oil conta- scale tests, Alaskan beaches received large
bioremediation almost as a last resort. mination. The potential for truly massive scale nutrient applications. Eventually,
Surface-water bioremediation technolo- spills from modern supertankers, and the about 110 miles of beach were treated with
gies are largely being developed in place. readily visible direct impact on affected many tons of nutrient materials. Both
An ongoing example is the General Electric areas, have captured the public's attention water soluble and oleophilic fertilizers were
(GE) site in Fort Edward, New York, on and sensitized regulatory and industry effective, with visible clearing of oiled
the upper Hudson River. For many years groups to the local destructive potential of beaches evident in many cases. The results
GE legally released PCBs into the river such accidents. Petroleum is a complex obtained indicate that, for the conditions
from a plant that manufactured capacitors. mixture of thousands of individual com- encountered, the bioremediative action of
When PCBs became priority environmen- pounds, and the degradation pathways of indigenous bacteria can safely be acceler-
tal pollutants in the early 1980s, at least 20 spilled oil are numerous and complex. ated 2- to 4-fold over control beaches by a
miles of the river bottom were found to be Biodegradation, especially by microbes, is single addition of nutrients. A second
contaminated downstream of the plant. believed to be one of the primary mecha- application 3 to 5 weeks later boosted this
GE began looking for remediation options. nisms of ultimate removal of petroleum figure to as high as 5- to 10-fold (116).
In 1991, GE conducted an extensive field hydrocarbons from marine and shore envi- Analysis of the process and steps involved
research program to characterize natural ronments (114,115). Acceleration of this in this bioremediation, and final assess-
degradation of PCBs at this site (112) and natural process is the objective of bioreme- ment, are still underway. Assessments of
discovered that the indigenous consortia of diation efforts. Bioremediation has yet to inoculation or seeding oil spills with
microorganisms was exceptionally good at become an established spill-response tech- selected microorganisms have, thus far,
degrading PCBs. Presumably, since the nology, but some attempts to implement it been inconclusive (118,120).
PCBs have been present in this site for a have been encouraging. The inability of The greatest theoretical problem with
significant time period (at least 35 years), established nonbiological techniques to bioremediation as a first response to oil
the indigenous microorganisms have cope with recent large spills has led to spills is the time of action. Movement of
adapted to utilize the material as a food increased interest in bioremediation. oil slicks toward sensitive areas (i.e., coastal
source. Both anaerobic and aerobic bio- Special problems associated with marine oil wetlands, beaches, shellfish beds, etc.) may
degradation have been identified as part of spills include the uncontained nature of the necessitate a rapid nonbiological response,
the natural process of remediation, which waste, the potential size of the contami- such as chemical dispersal or burning, to
can be slow. Field tests in cylindrical cais- nated area, and difficulty in access for prevent contamination. In such cases,
sons sunk into the river sediment at this remediative and monitoring activities. bioremediation may prove useful as a sec-
site have identified the variables that can be As with other forms of in situ bioreme- ondary treatment option or when paired
manipulated to enhance in situ biodegrada- diation, natural biodegradation of marine with nonbiological methods of degradation
tion of PCBs. The addition of inorganic oil spills may be enhanced by inducing enhancement. A proposed first response to
nutrients, the organic co-metabolite changes in either the microbial population oil spills utilizes titanium-dioxide coated
(biphenyl), and oxygen significantly or the availability of microbial nutrients. floating glass microbeads to catalyze pho-
increased PCB degradation rates. Addition Most researchers have concluded that tooxidation of the oil (119,120). When
of selected PCB-degrading bacterial cul- nutrient availability is the chief limitation these beads are applied to a floating slick, a
tures did not dramatically improve of natural biodegradation and most microfilm of oil coats the beads, and the
biodegradative efficiency. No more than research has been directed toward enhanc- semiconducting titanium dioxide compound
60% of the PCBs was degraded in any lab- ing nutrient availability (116). Marine oil- absorbs sunlight energy, thus catalyzing the
oratory or field experiments, a finding spill cleanups represent some of the largest oxidation of the organic microfilm (121).
attributed to tight sediment adsorption of in situ remediation projects ever attempted. The resulting breakdown products are more
the least water-soluble PCB compounds The March 1989 spill of 11 million gallons water soluble and more bioavailable, which
(113). More information on degradation of crude oil from the supertanker Exxon results in more rapid biodegradation.
rates, products, and variability under nat- Valdez into Prince William Sound, Alaska, Another emerging application of biore-
ural conditions is required for a realistic provided a testing ground for many nutri- mediation, with potential yet to be fully
evaluation of the role that bioremediation ent enrichment technologies. The U.S. realized, is biodegradation and/or removal
may play in this and other surface-water EPA and Exxon spent about $8 million on of environmentally undesirable compounds
sites contaminated by organic waste. a joint program to test and apply such mea- from air through biofilter technology. This
Humans have long exploited the vol- sures (117). Open-ocean nutrient enrich- technology has been shown, primarily by
ume-dilution power of the sea to dispose of ment is problematic. Physical dispersion of groups working in Europe, to be suitable
unwanted wastes. Although concern about oil and nutrients makes both application for large-scale biodegradation of VOCs
waste accumulation in marine environ- and assessment difficult. The impossibility [(122); AJ Dragt and SPP Ottengraf,
ments is increasing, especially for coastal of maintaining discrete treated and control unpublished data]. The idea is to direct the
waters, marine remediation efforts are areas of a floating slick makes any evalua- flow of a VOC-laden gas stream through a
nearly nonexistent. The notable exception tion of success suspect. In Prince William bed containing mixed cultures of microor-
to this rule is crude oil and refined petro- Sound, therefore, the beaches became the ganisms that mineralize the VOCs. The
leum product spills. Tanker spills account primary bioremediation target. The micro- actual filter bed is a wet, biologically active
for only 13% of the estimated 3.2 million organisms present in the beach sediment layer of compost, peat, or soil, which pro-
metric tons of annual marine petroleum were considered diversified and suffiently vides both a structural support matrix and
hydrocarbon inputs (114). Yet tanker spills capable to complete the oil biodegradation a source of inorganic nutrients to the micro-
have remained the focus of research efforts desired. Following the appraisal of small bial population. Inert structural additives,

14 Environmental Health Perspectives - Vol 105, Supplement * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

such as plastic beads, may be included to available. Some compounds, notably be expected to decrease as techniques and
decrease flow resistance and reduce struc- the organohalogens, are highly resistant to equipment are improved and become more
tural changes due to aging. Compost mix- aerobic biodegradation (termed recalcitrant standardized. Cost estimates for various
tures made from municipal wastes, wood or persistent wastes). Resistance of most technologies may be obtained from con-
chips, bark, or leaves have usually been the aromatic and aliphatic compounds to tractor-supplied values included in the U.S.
media used in European gas biofilters. U.S. degradation is dramatically increased by EPA Vendor Information System for Inno-
designs have typically utilized soil beds, halogenation (most commonly chlorina- vative Treatment Technologies database
which suffer from lower biodegradation tion); further halogenation results in (U.S. EPA, Research Triangle Park, NC).
capacity, larger space requirements, and increased resistance (102). Costs tend to be highly dependent on spe-
higher gas flow resistance (122). Given suf- Anaerobic microbes degrade organics cific properties of a given site, and so-called
ficient retention time in the filter, the reductively, eventually resulting in the typical values may not be meaningfully
organic contaminants diffuse into the mineral end product methane. In the case applied to specific case-cost projections.
aqueous biolayer (or biofilm) surrounding of carbohydrate compounds, carbon diox- Bioremediation often occurs through
the filter-bed particles, where they are ide and free hydrogen also are produced. unassisted natural biodegradation, albeit
degraded by microbial metabolism. The Although they are not usually utilized for slowly. If consideration of time scale were
final products are microbial biomass, routine waste degradation, some anaerobes eliminated, the "do nothing" approach
resulting from growth of the microorgan- are very adept at dechlorination of common might be best and has been suggested as
isms, and mineral end products. Volatile recalcitrant organochlorine compounds, the optimum overall remediation option in
end products will be carried out in the gas notably PCBs, organochlorine pesticides, certain types of contamination, such as
effluent stream. Nonvolatiles will accumu- such as DDT, and chlorinated aliphatics, marine oil spills (127). Even so, political
late in the filter media, which eventually such as the industrial solvent trichloroethy- and legal considerations often effectively
must be changed. Naturally occurring lene (TCE) (86,123,124). Thus anaerobic eliminate the "do nothing" option. How-
microorganisms are usually present in microbial catabolism (sometimes called fer- ever, cost factors are likely to result in the
quantities adequate to handle easily biode- mentation) offers a bioremediation option selection of something other than the
gradable compounds like alcohols, ethers, to deal with persistent wastes. Complete fastest option.
simple aromatics, etc. More degradation- anaerobic degradation of wastes, however, Risks to human or environmental health
resistant chemicals, such as nitrogen- and may be slow. associated with a particular remediation
sulfur-containing organics and especially The major problem with anaerobic scheme often play a major role in selection
chlorinated organics and aliphatics, may digestion of organochlorine wastes is that of the most desirable technique. Examples
require inoculation with selected strains of biodegradation is often incomplete (at least include the risk of waste dispersal, as when
microbes to achieve desired degradation on a practical time scale) and may result in surface spills threaten groundwater quality,
efficiencies. Although every application toxic metabolites. The use of mixed cul- the risk of transportation of toxic wastes
must be evaluated individually, biofilter tures containing both aerobes and anaer- through populated or environmentally sen-
technology represents a VOC abatement obes facilitates mineralization of many sitive areas, or the risk of accidental release
option that is competitive in many cases on organochlorines (125). In practice, a of genetically engineered organisms.
both an efficiency and a cost basis (122). Assequential bioreactor system utilizing both Appreciation of the potential of natural
many as 500 aerial biofilters may be func- anaerobic and aerobic reactors could be systems to regulate levels of aquatic toxi-
tioning in Germany and the Netherlands, employed. For example, PCBs or chlori- cants has led to the development of con-
ranging from small, simple systems associ- nated aromatics could be dechlorinated structed wetlands for bioremediation of
ated with farms and food-processing plants anaerobically, then fed into an aerobic complex wastes. It has been observed that
to large-scale facilities at chemical plants,bioreactor to be fully mineralized to carbon wetlands have a buffering ability on surface
foundries, print shops, and paint shops. dioxide and water. Similarly, TCE and per- waters with respect to circulating nutrient
Biodegradation efficiencies in excess of chloroethylene may be reductively metabo- and pollutant levels. Wetlands have the
90% have been achieved in a number of lized to vinyl chloride (a toxic chemical), capacity to store excess nutrients or wastes
industrial installations (AJ Dragt and SDP which can then be subjected to aerobic and to release stored excesses under the
Ottengraf, unpublished data). biodegradation. Commercial versions of right environmental conditions [for a col-
such two-stage hybrid bioreactor systems lection of reviews on this subject, see
Aerobic versus Anaerobic are currently under development (117). Hammer (128)]. A constructed wetland is
Metabolism of Wastes Isolation and characterization of dehaloge- an artificial habitat, most visibly made up
For purposes of bioremediation, aerobic nases (dehalogenating bacterial enzymes) of vascular plants and algal colonies, which
microbial metabolism has traditionally been for possible development of immobilized also provide a structural and nutritional
the focus of attention. Aerobic degradative enzyme reactors and biofilters are also support for an associated, highly heteroge-
pathways in microbes and in animals break being conducted (126). neous microbial community. One of the
down organic molecules oxidatively by most promising applications of constructed
using divalent oxygen or other active oxy- Counting Time, wetlands is for in situ bioremediation of
gen species, such as hydrogen peroxide, as Dollars, and Risks metal contamination. It is not always
electron acceptors. Aerobic catabolism of For a given remediation project, it is likely known to what extent the observed metal
organics ultimately results in familiar min- that several bioremediation options exist. removal in natural wetlands is due to bac-
eral products-carbon dioxide and water. The most obvious constraint on any reme- terial action and what is due to higher
Aerobes are capable of degrading most diation effort is cost; the business of waste plant or algal activity. In any case, many of
organic wastes, provided enough oxygen is remediation can be very costly. Costs may these organisms exist in a symbiotic

Environmental Health Perspectives * Vol 105, Supplement * February 1997 15

 BONAVENTURA AND JOHNSON

arrangement, and multitrophic cultured present in a given soil or sediment sample that innovative and novel processes for
systems are increasingly being viewed as an are in development (91). Gene probing dealing with complex waste mixtures be
alternative to monocultures or even hetero- can also give an indication of the relative explored. Nondegradable wastes in such
geneous bacterial cultures. Field tests on natural abundance of organisms with the mixtures, notably heavy metals, can be
acid mine drainage effluent have indicated potential to degrade specific pollutants at a extracted from mixed-waste streams by bio-
that such systems are capable of removing given site. chemical action and may be concentrated
metals via multiple pathway biological There is a clear need for improved by the method of bioaccumulation whereby
action (129). The use of both natural and methods of environmental surveillance for wastes are absorbed and stored in the tis-
constructed wetlands for heavy metal the prevention of adverse environmental sues of the organisms utilized. Improved
abatement is of great potential value, but conditions. Continued development of bioremediation of complex mixtures might
questions remain about the eventual fates new methods, including lab-bench assays take advantage of the fact that microbes
of the metals. Some means of extraction, and gene-probe technologies and their uti- can be selected to mobilize, immobilize, or
such as removal of plant or sediment mate- lization, may provide some of the desired fix compounds or ions in such a way that
rial, is necessary to prevent remobilization information and early warning for environ- they are rendered susceptible to further
of metals from dead organic material or mental hazards. When required, bioreme- treatment. The first stage of the process
trophic transfer to grazing animals. diative approaches need to be applied with may require the action of a biodegrading,
Aerial biofiltration is a developing tech- the understanding that each local environ- surfactant-producing or bioaccumulating
nology that has yet to see widespread appli- ment requires individual attention and organism. Development of practical means
cation in the United States. It has received detailed site evaluation. In bioremediation of exploiting such bioactivities under field
extensive attention in this review because it of a contaminated area, performance feed- conditions is a definite research need.
is the only proven biological system that back to researchers with regard to the Improved methods for evaluating the
can degrade airborne pollutants. At pre- transport, fate, and possible toxicity of the degree of pollutant biodegradation in com-
sent, it is an emission control technology metabolites produced is of tremendous plicated environmental settings need to be
applicable only to pollutant point sources. value for method refinement. Moreover, developed, with simpler, less expensive,
Whereas no remediation technique has yet the site evaluation processes must incorpo- and more rapid assay procedures. Although
been established to remove ambient levels rate expertise from those knowledgeable in complete mass-balance analyses are desir-
of organic pollutants from air, extensions other remediation technologies as well as able, they are seldom obtainable. In light
of biofilter technology may yield such tech- bioremediation experts. Coupled and inte- of this deficit, the durability and effective-
niques in the future. Potential technical grated methods of containment, destruc- ness of bioremediative technologies will
problems include maintenance of degrada- tion, and biodegradation of pollutants are require other indicators. New and better
tion efficiency with variable off-gas flow certain to yield more cost-effective cleanup models for the processes involved, as well
rates, accumulation of damaging metabolic solutions than procedures that focus on a as improved diagnostic assays, may be
by-products in the filter bed (inorganic single remediation technology. required. If adequate models are developed,
acids from sulfur, nitrogen, and chloride- The primary limitation to the widespread their use would greatly facilitate the design
containing wastes, for example), and the use of many bioremediation approaches is of cost-effective, safe, and practical biore-
possibility of release to the atmosphere of often the extent to which the pollutant is mediative technologies to be applied in
undesirable amounts of microbial or fungal available to the microbial population. The large-scale operations.
organisms or spores. bioavailability of many chemicals dimin-
ishes with time as a result of weathering Conclusions
Environmental Diagnostics and aging phenomena, and the time win- Effective bioremediation can be advanced
and Surveillance Needs dow in which appropriate bioremediation and facilitated by making products that are
Bioremediation is an emerging field, the technologies can be employed requires fur- readily susceptible to biodegradation.
full potential of which is as yet unknown. ther definition. Many organic pollutants Keeping the principles of bioremediation
There is a tremendous need for further do not readily enter the bioactive, aqueous in mind during product and process devel-
basic research and development, especially phase of soil and sediment environments. opment is both prudent and profitable in
in the areas of environmental site and waste Their bioavailability to the microbial pop- the long term, as 3M (St. Paul, MN) and
diagnostics, waste-technology matching, ulation might be appreciably increased by other leading international companies have
and integration of multiple remediation the use of appropriate surfactants, disper- shown (131). Unfortunately, long-term
techniques. sants, chelators, or emulsifiers. The physi- planning does not come naturally to every-
There are two methods traditionally cal matrix in which pollutants are found one and there is great resistance to environ-
used to monitor bioremediation. The most largely determines the rate at which the mental protection and cleanup because of
common approach monitors pollutant and pollutants become bioavailable. Recognized the effort and expense involved (132).
metabolite concentrations as an indirect areas for research advancement include Bioremediation is a technological attempt
measure of the extent of biological activity alteration of sorption and desorption kinet- to exploit the abilities of microbes and
that has occurred. The more rigorous ics at contaminated sites to enhance the other members of the biosphere to restore
approach is to directly monitor the bacter- rate of bioremediation. Improved methods and maintain environmental quality for all
ial population. Modern methods of molec- to make pollutants more bioavailable and forms of life in the ecosystem, especially
ular biology offer alternatives to the tedious an increased knowledge of the critical para- humans. Education is important in achiev-
and time-consuming classical culture-and- meters involved are clearly needed. ing the widespread practices of prevention,
identify methods. Gene-probe methods A major recommendation of a U.S. recycling, and remediation for the purpose
that directly identify the microbial species EPA bioremediation workshop (130) was of improving future environmental health

16 Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

and quality of life. Through education, efforts are as yet incomplete, and some generated contaminants are also adversely
societal customs can change in ways that efforts have had little effect. In some cases, affecting the health of undetermined num-
both reduce and recycle wastes. costs are astronomical, e.g., $1 billion at bers of people. It is clear that new bioreme-
Perhaps the larger problem facing pol- the Rocky Mountain Arsenal near Denver, diation technologies that can better
icy makers in the future is how to decide Colorado (136). Although there are cases monitor and control many types of societal
where available bioremedial dollars will where human health has been a legitimate wastes are emerging. However, there is lit-
benefit human and environmental health concern (137), most cleanup decisions tle incentive to find and develop new tools
the most. For instance, in the last 15 years, appear to be made in the absence of any while relatively inexpensive chemical and
more than 40,000 hazardous waste sites evidence of adverse human effects (138). bioremediation technologies that are
have been identified in the United States Although the issues involved are unde- proven and effective are unused. Water
alone, but remedial efforts have been niably complex, a considerably improved quality, which could benefit by new and
undertaken at only a few hundred sites and and enlarged remedial campaign would existing bioremediative methods, can only
are largely restricted to the approximately seem necessary to deal with all identified get worse as populations grow. Priority set-
1300 sites on National Priority List toxic waste sites in an adequate, responsible, ting, clearly stated environmental policies,
(Superfund sites) (133,134). Target and expeditious fashion. In the meantime, and the implementation of appropriate
cleanup goals have been judged to be ineffective sewage treatment plants, septic remediative measures to deal with our con-
highly unrealistic in some cases (i.e., tanks, and improper methods for dealing taminated land and water resources are
exceeding a 10,000-fold safety factor) with farm animal waste are also of great badly needed.
(135). At most of these sites remedial practical concern; and these biologically

REFERENCES
1. Carson R. Silent Spring. New York:Fawcett Crest, 1962. 17. Ember L. Toxic release inventory data show steady drop in
2. Bonaventura C, Bonaventura J, Bodishbaugh DF. emissions. Chem Eng News 70:13-15 (1992).
Environmental remediation: approaches and processes. In: 18. Barker JC, Zublena JP. Livestock manure nutrient assessment
Ecotoxicity and Human Health, A Biological Approach to in North Carolina. In: Proceedings of the 7th International
Environmental Remediation (de Serres FJ, Bloom AD, eds). Symposium on Agricultural and Food Processing Wastes. St.
Boca Raton, FL:CRC Press, 1996; 183-210. Joseph, MI:American Society of Agricultural Engineers
3 Bonaventura C, Bonaventura J, Bodishbaugh DF. 1995;98-106.
Environmental remediation: applications and new horizons. In: 19. North Carolina Coastal Federation. State of the Coast Reports.
Ecotoxicity and Human Health, A Biological Approach to Coastal Review. Special Edition. Newport, NC:North Carolina
Environmental Remediation (de Serres FJ, Bloom AD, eds). Coastal Federation, 1996.
Boca Raton, FL:CRC Press, 1996;211-242. 20. Engel DW, Noga EJ. Shell disease in the blue crabs of the
4. Chaudhry GR, ed. Biological Degradation and Bioremediation Pamlico River. Environs X:3-5 (1989).
ofToxic Chemicals. Portland, OR:Dioscorides Press, 1994. 21. Culotta E. Red menace in the world's oceans. Science
5. Laws, E. Aquatic Pollution. An Introductory Text. 2nd ed. 257:1476-1477 (1992).
New York:Wiley & Sons, 1993. 22. Paerl HW. Dynamics of Blue-Green Algal Blooms in the
6. Sayler GS, Fox R, Blackburn JW, eds. Environmental Lower Neuse River, North Carolina: Causative Factors and
Biotechnology for Waste Treatment. New York:Plenum Press, Potential Controls. Rpt No 229. Raleigh, NC:Water Resources
1991. Research Institute, North Carolina State University (1987).
7. Weber WJ Jr, DiGiano FA. Process Dynamics in 23. Paerl HW, Mallin MA, Donahue CA, Go M, Peleris BL.
Environmental Systems. New York:Wiley & Sons, 1996. Nitrogen Loading Sources and Eutrophication of the Neuse
8. Bowen VT, Rubinson AC. Uptake of lanthanum by a yeast. River Estuary, NC: Direct and Indirect Roles of Atmospheric
Nature 167:1032 (1981). Deposition. Rpt 291. Raleigh, NC:Water Resources Research
9. Mann H, Fyfe WS. Algal uptake of uranium and some other Institute, North Carolina State University (1995).
metals: implications for global geochemical cycling. 24. National Oceanic and Atmospheric Administration. Gulf of
Precambrian Res 30:337-349 (1985). Mexico "Dead Zone." News Release. Washington:National
10. Kuyucak N, Volesky B. Accumulation of cobalt by marine Oceanic and Atmospheric Administration, November 17, 1995.
algae. Biotechnol Bioeng 33:809-814 (1989). 25. Murray CJL, Lopez AD. Evidence-based health policy-lessons
11. Kuyucak N, Volesky B. Desorption of cobalt-laden algal bio- from the Global Burden of Disease Study. Science 274:740-743
mass. Biotechnol Bioeng 33:815-822 (1989). (1996).
12. Ashford LS, Noble JA. Population policy: consensus and chal- 26. Ellis KV. The tertiary treatment of sewages. Effluent Water
lenges. Consequences 2:25-36 (1996). TreatJ 20:422-430 (1980).
13. Brown P. Race, class, and environmental health: a review and 27. Ryther JH, Dunstan WM. Nitrogen, phosphorus, and
systematization of the literature. Environ Res 69:15-30 (1995). eutrophication in the coastal marine environment. Science
14. Marth E, Sixl W, Bencko V, Medwed M, Lapajne S, Voncina 171:1008-1013 (1971).
E, Brumen S. People on the garbage dumps of Cairo: a toxico- 28. Schindler DW. Eutrophication and recovery in experimental
logical in vivo model? Cent Eur J Public Health 3:154-157 lakes: implications for lake management. Science 164:897-899
(1995). (1974).
15. Hoffmann GR. Mutagenicity testing in environmental toxicol- 29. Eliassen R, Tchobanoglous G. Removal of nitrogen and phos-
ogy. Environ Sci Technol 16:560A-574A (1982). phorus from waste water. Environ Sci Technol 3:536-541
16. Zeeman M, Auer CM, Clements RG, Nabholz JV, Boethling (1969).
RS. U.S. EPA regulatory perspectives on the use of QSAR for 30. Pressley TA, Bishop DF, Roan SG. Ammonia-nitrogen
new and existing chemical evaluations. SAR QSAR Environ removal by breakpoint chlorination. Environ Sci Technol
Res 3:179-201 (1995). 6:622-628 (1972).

Environmental Health Perspectives * Vol 105, Supplement * February 1997 17

 BONAVENTURA AND JOHNSON

31. Fertik HA, Sharpe R. Optimizing the computer control of 55. Galun M, Keller P, Feldstein H, Galun E, Siegel S, Siegel B.
breakpoint chlorination. Instrum Soc Am Trans 19:3-17 Recovery of uranium (VI) from solution using fungi. Water Air
(1980). Soil Pollut 20:277-285 (1983).
32. Allen J. Sewage farming. Environment 15:36-41 (1973). 56. Tsezos M. The role of chitin in uranium adsorption by
33. Page AL, Gleason TL III, Smith JE Jr, Iskandar IK, Sommers Rhizopus arrhizus. Biotechnol Bioeng 25:2025-2040 (1983).
LE. Utilization of municipal wastewater and sludge on land. In: 57. Yakubu NA, Dudeney AWL. Biosorption of uranium with
Proceedings of the 1983 Workshop. EPA-600/S9-84-003. Aspergillus niger. In: Immobilisation of Ions by Biosorption
Washington:U.S. Environmental Protection Agency, 1984. (Eccles H, Hunt S, eds). Chichester, England:Ellis Horwood,
34. Payne JF, Overman AR. Performance and long-term effects of 1986.
a wastewater spray irrigation system in Tallahassee, Florida. In: 58. Nakajima A, Sakaguchi T. Recovery of uranium by tannin
Report for Wastewater Division Underground Utilities. immobilized on matrices which have an amino group. J Chem
Ta lahassee, FL:City of Tallahassee, 1987. Technol Biotechnol 47:31-38 (1990).
35. Allhands MN, Overman AR. Effects of municipal effluent irri- 59. North Carolina Department of Environment, Health, and
gation on agricultural production and environmental quality. Natural Resources. Air Quality Trends Summary 1972-1989.
In: Report or Wastewater Operations. Tallahassee, FL:Water Raleigh, NC:NC Division of Environmental Management,
& Sewer Department of Tallahassee, 1989. 1991.
36. van Mannen JMS, Welle IJ, Hageman G, Dallinga JW, 60. Slemr F, Langer E. Increase in global atmospheric concentra-
Mertens PLJM, Kleinjans JCS. Nitrate contamination of drink- tions of mercury inferred from measurements over the Atlantic
ing water: relationship with HPRT variant frequency in lym- Ocean. Nature 355:434-437 (1992).
phocyte DNA and urinary excretion of N-nitrosamines. 61. Swain EB, Engstrom DR, Brigham ME, Henning TA,
Environ Health Perspect 104:522-528 (1996). Brezonik PL. Increasing rates of atmospheric mercury leposi-
37. Xu G, Song P, Reed PI. The relationship between gastric tion in midcontinental North America. Science 257:784-787
mucosal changes and nitrate intake via drinking water in a (1992).
high-risk population for gastric cancer in Moping country, 62. Nakagawa R. Studies on the levels in atmospheric concentra-
China. Eur J Cancer Prev 1:437-443 (1992). tions of mercury in Japan. Chemosphere 31:2669-2676
38. Garry VF, Schreinemachers D, Harkins ME, Griffith J. (1995).
Pesticide appliers, biocides and birth defects in rural 63. Burge P, Evans S. Mercury contamination in Arkansas game-
Minnesota. Environ Health Perspect 104:394-399 (1996). fish. A public health perspective. J Ark Med Soc 90:542-544
39. McGrath SP, Chaudri AM, Giller KE. Long-term effects of (1994).
metals in sewage sludge on soils, microorganisms and plants. J 64. Osowski SL, Brewer LW, Baker OE, Cobb GP. The decline of
Ind Microbiol 14:94-104 (1995). mink in Georgia, North Carolina, and South Carolina: the role
40. Sopper WE, Kerr SN. Revegetating strip-mined land with of contaminants. Arch Environ Contam Toxicol 29:418-423
municipal sewage sludge. EPA-600/S2-84-81-182. Cincinnati, (1995).
OH:U.S. Environmental Protection Agency, 1981. 65. Koli AK, Williams WR, McClary EB, Wright EL, Burrell TM.
41. So pper WE, Seaker EM. Strip mine reclamation with munici- Mercury levels in freshwater fish of the state of South Carolina.
pal sludge. EPA-600/S2-84-035. Cincinnati, OH:US Bull Environ Contam Toxicol 17:82-89 (1977).
Environmental Protection Agency, 1984. 66. Lange TR, Royals HE, Connor LL. Mercury accumulation in
42. Outwater A. Water. New York:Harper Collins, 1996. largemouth bass (Micropterus salmoides) in a Florida lake. Arch
43. Patrick RE, Ford E, Quarles J. Groundwater contamination in Environ Contam Toxicol 27:466-471 (1994).
the United States. 2nd ed. Philadelphia:University of 67. Giesy JP, Verbrugge DA, Othout RA, Bowerman WW, Mora
Pennsylvania Press, 1987. MA, Jones PD, Newsted JL, Vandervoort C, Heaton SN,
44. U.S. EPA. Groundwater Protection. Washington:U.S. Aulerich RJ, et al. Contaminants in fishes from Great Lakes-
Environmental Protection Agency, 1980. influenced sections and above dams of three Michigan rivers. I,
45. Deportes I, Benoit-Guyod JL, Zmirou D. Hazard to man and Concentrations of organochlorine insecticides, polychlorinated
the environment posed by the use of urban waste compost: a biphenyls, dioxin equivalents, and mercury. Arch Environ
review. Sci Total Environ 172:197-22 (1995). Contam Toxicol 27:202-212 (1994).
46. Sawyer TK, Nerad TA, Gaines J, Small EB, Cooper RA. 68. Peterson DE, Kanarek MS, Kuykendall MA, Diedrich JM,
Bacterial and protozoan indicators of sewage contamination of Anderson HA, Remington PL, Sheffy TB. Fish consumption
deepwater sediments at Hudson Canyon. J Mar Environ Eng patterns and blood mercury levels in Wisconsin Chippewa
2:369-376 (1996). Indians. Arch Environ Health 49:53-58 (1994).
47. Narang H. Origin and implications of bovine spongiform 69. Zajic J E. Microbial Biogeochemistry. New York:Academic
encephalopathy. Proc Soc Exp Biol Med 211:306-322 (1996). Press, 1969.
48. Centers for Disease Control and Prevention. World Health 70. Antonovics J, Bradshaw AD, Turner RG. Heavy metal toler-
Organization Consultation on public health issues related to ance in plants. Adv Ecol Res 7:1-85 (1971).
bovine spongiform encephalopathy and the emergence of a new 71. Baker AJM, Brooks RR. Terrestrial higher plants which hyper-
variant of Creutzfeldt-Jakob disease. JAMA 275:1305-1306 accumulate metallic elements-a review of their distribution,
(1996). ecology and phytochemistry. Biorecovery 1:81-126 (1989).
49. Beran GW, ed. Handbook of Zoonoses. 2nd ed. Boca Raton, 72. Shann JR. The role of plants and plant/microbial systems in
FL:CRC Press, 1994. the reduction of exposure. Environ Health Perspect 103(Suppl
50. Hugh-Jones ME, Hubbert WT, Hagstaad HV. Zoonoses. 5):13-15 (1995).
Ames, IA:Iowa State University Press, 1995. 73. Rugh CL, Wilde HD, Stack NM, Thompson DM, Summers
51. Dismukes WE. Emerging and reemerging infections. Am J AO, Meagher RB. Mercuric ion reduction and resistance in
Med 100:12-14 (1996). transgenic Arabidopsis thaliana plants expressing a modified
52. Tenover FC, McGowan JE Jr. Reasons for the emergence of bacterial merA gene. Proc Natl Acad Sci USA 93:3182-3187
antibiotic resistance. Am J Med Sci 311:9-16 (1996). (1996).
53. Tenover FC 1995. The best of times, the worst of times. The 74. Neilands JB. Microbial iron compounds. Annu Rev Biochem
global challenge of antimicrobial resistance. Pharm World Sci 50:715-71 (1981).
17: 149-151. 75. DeVoe IW, Holbein BE. A new generation of solid-state metal
54. Tolley MR, Macaskie LE. The use of bioreactor kinetics to complexing materials, models and insights derived from biolog-
quantify the effect of interfering agents on bioreactor efficiency: ical systems. In: Trace Metal Removal from Aqueous Solutions.
'proof of principle' using uranium-accumulating Citrobacter sp. Special Publication 61. (Thompson R, ed). London:Royal
in a plug-flow reactor. Biotechnol Tech 4:83-88 (1990). Society of Chemistry, 1986.

18 Environmental Health Perspectives * Vol 105, Supplement 1 * February 1997

 HEALTHY ENVIRONMENT FOR HEALTHY PEOPLE

76. Premuzic ET, Francis AJ, Lin M, Schubert J. Induced forma- 97. Gibson DJ. Eugene W. Caldwell Lecture. Technology: the key
tion of chelating agents by Pseudomonas aeruginasa grown in to controlling health care costs in the future. Am J Roentgenol
the presence of uranium and thorium. Arch Environ Contam 163:1289-1293 (1994).
Toxicol 14:759-768 (1985). 98. Warford JJ. Environment, health, and sustainable develop-
77. Macaskie LE. The application of biotechnology to the treat- ment: the role of economic instruments and policies. Bull
ment of wastes produced from the nuclear fuel cycle: biodegre- World Health Organ 73:387-395 (1995).
dation and bioaccumulation as a means of treating 99. Carberry JB, Benzing TM. Peroxide pre-oxidation of recalci-
radionuclide-containing streams. Crit Rev Biotechnol trant toxic waste to enhance biodegradation. Water Sci
11:41-112 (1991). Technol 23:367-376 (1991).
78. Macaskie LE, Dean ACR. Metal sequestering biochemicals. In: 100. Bachofen R, ed. Microorganisms in nuclear waste disposal, Part
Biosorption of Heavy Metals (Volesky B, ed). Boca Raton, I. Experientia 46:777-851 (1990).
FL:CRC Press, 1990;199-248. 101. Bachofen R, ed. Microorganisms in nuclear waste disposal, Part
79. Mergeay M. Towards an understanding of the genetics of bac- II. Experientia 47:507-592 (1991).
terin metal resistance. Trends Biotechnol 9:17-24 (1991). 102. Stroo HF. Biotechnology and hazardous waste treatment. J
80. Appanna VD, Gazso LG, Huang J, St. Pierre M. A microbial Environ Qual 21:167-175 (1992).
model for caesium containment. Microbios 86:121-126 103. U.S. EPA. Slurry Biodegradation. EPA/540/2-90/016.
(1996). Washington:U.S. Environmental Protection Agency, 1990.
81. Brierley JA, Luinstra L. Biooxidation-heap concept for pretreat- 104. Middleton AC, Nakles DV, Linz DG. The influence of soil
ment of refractory gold ore. In: Biohydrometal1urgical composition on bioremediation of PAH-contaminated soils.
Technologies, Vol 1 (Toma AE, Wey JE, Lakshmanan VL, Remediation, Autumn;391-406 (1991).
eds). Warrendale, PA:The Minerals, Metals & Materials 105. Sims JL, Sims RC, Matthews JE. Approach to bioremediation
Society, 1993;437-448. of contaminated soil. Hazard Waste Hazard Mater 7:117-149
82. Moffat AS. Microbial mining boosts the environment, bottom (1990).
line. Science 264:778 (1994). 106. Goldstein RM, Mallory LM, Alexander M. Reasons for possi-
83. Merson J. Mining with microbes. New Scientist 133:17-19 ble failure of inoculation to enhance biodegradation. Appl
(1992). Environ Microbiol 50:977-983 (1985).
84. The Netherlands Organization for Applied Scientific Research. 107. Ramadan MA, El-Tayeb OM, Alexander M. Inoculum size as a
Getting bugs to remove heavy metals from bulk wastes. factor limiting success of inoculation for biodegradation. Appl
Applied Research, TNO Newsletter 38:1 (1991). Environ Microbiol 56:1392-1396 (1990).
85. Cerniglia CE, Heitkamp MA. Microbial degradation of poly- 108. Foght JM, Westlake DWS, Johnson WM, Ridgway HF.
cyclic aromatic hydrocarbons (PAHs) in the aquatic environ- Environmental gasoline-utilizing isolates and clinical isolates of
ment. In: Metabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas aeruginosa are taxonomically indistinguishable by
the Aquatic Environment (Varanasi U, ed). Boca Raton, chemotaxonomic and molecular techniques. Microbiology UK
FL:CRC Press, 1989; 41-68. 142:2333-2340 (1996).
86. Abramowicz DA. Aerobic and anaerobic biodegradation of 109. Romeyer FM, Jacobs FA, Masson L, Hanna Z, Brousseau R.
PCBs: a review. Crit Rev Biotechnol 10:241-251 (1990). Bioaccumulation of heavy metals in Escherichia coli expressing
87. Bedard DL. Bacterial transformation of polychlorinated an inducible synthetic human metallothionein gene. J
biphenyls. In: Biotechnology and Biodegradation, Advances in Biotechnol 8:207-220 (1988).
Applied Biotechnology Series (Kamely D, Chakrabarty A, 110. Travis CC. Toxic waste in ground-water: can it be removed? J
Omenn GS, eds). The Woodlands, TX:Portfolio Pub Co, NIH Res 4:49-51 (1992).
1990;369-387. 111. Carroquino MJ, Gersberg RM, Dawsey WJ, Bradley MD.
88. MacRae IC. Microbial metabolism of pesticides and struc- Toxicity reduction associated with bioremediation of gasoline-
turally related compounds. Rev Environ Contam Toxicol contaminated groundwaters. Bull Environ Contam Toxicol
109:1-87 (1989). 49:224-231 (1992).
89. Thorn PM, Kreitinger JP, Stroo HF. Biodegradation of pesti- 112. General Electric Company. 1991 In Situ Hudson River Research
cides in soil. Presented at 84th Annual Meeting, Air and Waste Study: A Field Study on Biodegradation of PCBs in Hudson
Management Association, Vancouver, BC, Canada, June River Sediment. Final Report. Schenectady, NY:General Electric
16-21, 1991. In: Waste Treatment Technologies, Vol 11. Company, 1992.
Papers from the 84th Annual Meeting. 91-22.13. Pittsburgh, 113. Harkness MR, McDermott JB, Abramowicz DA, Salvo JJ,
PA:Air & Waste Management Association, 1991. Flanagan WP, Stephens ML, Mondello FJ, May RJ, Lobos JH,
90. Krieger J. Technique identifies bacteria that degrade wastes. Carroll KM, et al. In situ stimulation of aerobic PCB biodegrada-
Chem Eng News 70:36 (1992). tion in Hudson River sediments. Science 259:503-507 (1993).
91. Olson BH. Tracking and using genes in the environment. 114. National Research Council. Oil in the Sea: Inputs, Fates, and
Environ Sci Technol 25:604-611 (1991). Effects. Washington:National Academy Press, 1985.
92. Rambeloarisoa E, Rontani JF, Giusti G, Duvnjak Z, Bertrand 115. Atlas RM. Biodegradation of hydrocarbons in the environment.
JC. Degradation of crude oil by a mixed population of bacteria In: Environmental Biotechnology (Omenn GS, ed). New
isolated from sea-surface foams. Mar Biol 83:69-81 (1984). York:Plenum Press, 1988.
93. Aelion CM, Bradley PM. Aerobic biodegradation potential of 116. Office of Technology Assessment, U.S. Congress. Bioremediation
subsurface microorganisms from a jet fuel-contaminated for Marine Oil Spills-Background Paper. OTA-BP-0-70.
aquifer. Appl Environ Microbiol 57:57-63 (1991). Washington:U.S. Government Printing Office, 1991.
94. Criddle CS, DeWitt JT, Grbic-Galic D, McCarty PL. 117. Thayer AM. Bioremediation: Innovative technology for clean-
Transformation of carbon tetrachloride by Pseudomonas sp. ing un hazardous waste. Chem Eng News 69:23-44 (1991).
strain KC under denitrification conditions. Appl Environ 118. Pritchard PH, Costa CF. EPA Alaska oil spill bioremediation
Microbiol 56:3240-3246 (1990). project. Environ Sci Technol 25:372-379 (1991).
95. Henry SM, Grbic-Galic D. Influence of endogenous and 119. Jackson NB, Wang CM, Luo Z, Schwitzgebel J, Ekerdt JG,
exogenous electron donors and trichloroethylene oxidation tox- Brock JR, Heller A. Attachment of TiO2 powders to hollow
icity on trichloroethylene oxidation by methanotrophic cultures glass microbeads: activity of the TiO2-coated beads in the pho-
from a groundwater aquifer. Appl Environ Microbiol toassisted oxidation of ethanol to acetaldehyde. J Electrochem
57:236-244 (1991). Soc 138:3660-3664 (1991).
96. Alpert S. The Application of Molecular Genetics Techniques to 120. Rosenberg I, Brock JR, Heller A. Collection optics of TiO2
Bioremediation Processes. EPRI GS-6591, Project 2655-28. photocatalyst on hollow glass microbeads floating on oil slicks.
Palo Alto, CA:Electric Power Research Institute, 1989. J Phys Chem 96:3423-3428 (1992).

Environmental Health Perspectives * Vol 105, Supplement I * February 1997 19

 BONAVENTURA AND JOHNSON

121. Gerischer H, Heller A. Photocatalytic oxidation of organic 130. U.S. EPA. Summary Report: High-Priority Research Needs
molecules on TiO2 particles by sunlight in aerated water. J Workshop, Report of EPA Sponsored Workshop held April
Electrochem Soc 139:113-118 (1992). 15-16, 1991, Washington:U.S. Environmental Protection
122. Leson G, Winer AM. Biofiltration: an innovative air pollution Agency, 1991.
control technology for VOC emissions. J Air Waste Manag 131. Ottman JA. Green Marketing. Lincolnwood, IL: NTC
Assoc 41:1045-1054 (1991). Business Books, 1993.
123. Long-de Valliere CL, Petrozzi S, Zurrer D, Baier U, Dunn IJ. 132. Russ T. Voters sour on pro-Everglades sugar tax. In: Outside
Methods of anaerobic degradation of toxic compounds in Online [http://www.uvm.edu/bxli/reference/ata.html]
chemical and industrial wastes. In: Biological Waste Bellevue, WA:Starwaver Corp, November 15, 1996.
Treatment. New York:Alan R. Liss, 1989; 35-72. 133. Gochfeld M, Burger J. Investigations and activities at super-
124. Neilson AH. The biodegradation of halogenated organic com- fund sites. Occup Med 5:127-142 (1990).
pounds. J AppI Bacteriol 69:445-470 (1990). 134. Johnson BL. Nature, extent, and impact of Superfund haz-
125. Rochkind ML, Blackburn JW, Sayler GS. Microbial ardous waste sites. Chemosphere 31:2415-2428 (1995).
Decomposition of Chlorinated Aromatic Compounds. EPA 135. Stohrer G. Realistic risk assessment. Regul Toxicol Pharmacol
600/2-86/090. Cincinnati, OH:U.S. Environmental Protection 22:118-121 (1995).
Agency, 1986. 136. Anonymous. Costs set for arsenal plan. Eng News Rec 220:11
126. Janssen DB, Pentenga M, Van der Ploeg J, Pries F, Van der (1988).
Waarde J, Wonink E, Van den Wijngaard AJ. Environmental 137. Neuberger JS, Mulhall M, Pomatto MC, Sheverbush J,
biotechnology: Transformations of synthetic halogenated com- Hassanein RS. Health problems in Galena, Kansas: a heavy
pounds by microbial cultures and enzymes. Biomolecular Study metal mining Superfund site. Sci Total Environ 94:261-272
Center Annual Report. Groningen, The Netherlands: (1990).
Groningen University, 1990;65-67. 138. Doty CB, Travis CC. The Superfund remedial action decision
127. National Research Council. Using Oil Spill Dispersants on the process: a review of fifty records of decision. J Air Pollut Cont
Sea. Washington:National Academy Press, 1989. Assoc 39:1535-1543 (1989).
128. Hammer DA, ed. Constructed Wetlands for Wastewater 139. Satake K, Miyasaka K. Evidence of high mercury accumulation
Treatment: Municipal, Industrial and Agricultural. Chelsea, in the cell wall of the liverwort Jungermannia vulcanicola Steph.
MI:Lewis Publishers, 1989. to form particles of mercury-su[phur compounds. J Bryol
129. Batal W, Laudon LS, Wildeman TR. Bacteriological tests from 13:101-105 (1984).
the constructed wetland of the Big Five Tunnel, Idaho Springs, 140. Samecka-Cymerman A, Kempers AJ. Preliminary investigations
Colorado. In: Constructed Wetlands for Wastewater into the bioaccumulation of mercury by the liverwort Scapania
Treatment: Municipal, Industrial and Agricultural (Hammer undulata L. (Dum). Ecotoxicol Environ Saf31:57-61 (1995).
DA, ed). Chelsea, MI:Lewis Publishers, 1989; 550-557.

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