LIMITING FACTORS FOR THE
BIOREMEDIATIONOF C 0 N " A ' I X D
SOILS AND ORGANIC
WASTES
William Ney Hansard
Enviromental Management, Inc.
151 Brentwood Square
Nashville, Tennessee 37211
(615)834-8257
Many soils and wastes contaminated with biodegradable organic compounds
can be effectively treated using bioremediation technologies. Bioremediation,
in many cases, is more cost effective than traditional incineration, physical,
or chemical treatment technologies. By utilizing established biological
principles, the kinetics and effectiveness of biodegradation processes can be
significantly enhanced.
Even bioresistant compounds and some
concentrated organic chemical sludges can be biodegraded in special
reactors in which environmental conditions for chemical breakdown,
metabolic assimilation and cellular synthesis are optimized. By addressing
Liebig's Law of the Minimum limiting factors and providing a n environment
conditioned to promote cellular metabolism, bioremediation technology is
able to effectively treat a wide range of chemical contaminants.
INTRODUCTION
Bioremediation of contaminated soil and groundwater, and biotreatment of
industrial wastes is becoming an increasingly popular environmental
control technology. Until recently, biochemical treatment of wastes has been
largely restricted t o domestic and industrial wastewaters in this country.
Also, until just a few years ago, the USEPA and state regulatory agencies
have been reluctant t o approve the use of biotechnology in contaminated site
remediation projects. Today, some corporate and plant environmental
managers continue t o view the use of biotechnology with suspicion, as it is
not a well established and proven alternative for the treatment of complex
and toxic waste mixtures. This situation is improving steadily, however, and
bioremediation technology is gaining wider acceptance as a useful and
practical alternative t o traditional remediation and treatment technologies.
The USEPA has launched the Bioremediation Field Initiative to evaluate the
use of various biotechnology alternatives at contaminated sites. Over 140 pilot
scale and full scale studies are currently planned or in operation for
CERCLA/RCRA/UST sites [ll. The information developed will be used t o
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evaluate the performance of selected field applications, provide technical
assistance t o USEPA and state personnel with technical assistance in
evaluating biotechnology alternatives, and develop a treatability database
which will be available to the public through the Alternative Treatment
Technologies Information Center (ATTIC).
The USEPA launched the Superfund Technical Support Project (TSP) in 1988
t o provide regulatory personnel with technical assistance in evaluating
remedial alternatives for superhnd sites. During fiscal years 1988-1990, over
half of TSP’s requests for assistance have been for the evaluation of biological
technologies.
Additionally, the EPA and the University of Pittsburg Trust have formed the
National Environmental Technology Applications Corporation (NETAC), a
nonprofit corporation established in 1988 to participate in the development of
new bioremediation protocols. In 1990 the EPA formed the Bioremediation
Action Committee (BAC) t o facilitate the safe use of bioremediation
technologies to solve some of the nation’s pollution problems. The BAC is
divided into six subcommittees: Pollution Prevention, Oil Spill Response,
Research, Treatability Protocol, Education, and Data ID and Collection. All
of the subcommittees report t o the assistant administrator of the USEPA’s
Office of Research and Development, who functions as chair of the BAC.
BAC subcommittee members are represented by industry and academia, as
well as regulatory agencies.
At Purdue University’s 1991 Industrial Waste Conference, the keynote
speaker, Ms. Rita Colwell stated that she believed that the use of biological
technologies and the growth in environmental biotechnology industries
would exceed all current estimates and projections. Ms. Colwell is a
professor of microbiology at the University of Maryland in College Park,
Maryland, and Director of the Maryland Biotechnology Institute.
The current optimism for environmental biotechnologies is well founded.
There are, however, some potential problems associated with the misuse of
biotechnology. Misleading claims are made by some involved in the industry,
resulting in the loss of confidence in biotechnology. Probably a more
important problem is the use of biotechnology in inefficient or improper
applications, due to a lack of understanding of the limits of biotechnology.
Biotechnologies have many limitations, depending on the technology and
application under consideration. All biotechnologies have one factor in
common: all depend on functional biochemical mechanisms in order to
work. It has long been established that all living organisms are subject to
Liebig’s Law of the Minimum which states that metabolic activity and
growth are restricted by that environmental requirement or nutrient that is
available in the minimum quantity.
This paper will explore limiting factors that affect cellular metabolism and
the effectiveness and efficiency of biological technologies used i n
environmental applications. Limiting factors can be generally classified as
falling under three headings: 1) Substrate Limiting Factors, 2)
457
Environmental Limiting Factors, and 3) Biological Consortia Limiting
Factors. Much of the data concerning limiting factors has been derived from
the literature and the author’s experience. Since there is very limited data
available for biological reactors treating contaminated ground water, soils
and hazardous wastes, data for conventional, completely mixed activated
sludge reactors has been used to illustrate the concept of limiting factors and
to provide a baseline for evaluating them. Data values, therefore, presented
in this paper should be used as guidelines only. Treatability studies should
be employed to assess limiting factors on an application specific basis.
SUBSTRATELIMITING FACTORS
The term substrate defines the food source that is presented t o
microorganisms for degradation. For the purpose of this discussion organic
chemical compounds are defined as substrate. Organic substrate must be
biodegradable for biotechnology to be of any use. Some compounds, such as
PCBs, are extremely bioresistant, but recent research has shown that certain
organisms, such as the white rot fungus, may be able t o successfully degrade
PCBs. Toxicity is another important limiting factor. Microorganisms can be
sensitive t o both organic and inorganic toxic chemicals. In some cases
toxicity can be controlled or completely overcome by waste dilution,
pretreatment, carbon addition and environmental controls in the bioreactor.
By carehlly regulating the addition of substrate to the appropriate biological
reactors and monitoring and adjusting the reactor environment, problems
associated with substrate limiting factors can often be controlled.
Many classes of organic compounds are or may be suitable for
biodegradation. Table 1 identifies some classes of chemicals that may be so
considered [21.
458
TABLE 1
Classes of Chemicals that May Be Suitable for Bioremediation
Using
Aerobic
Biodegradation
Process
Class
Examunle
Monochlorinatednzene
aromatic
compounds and
Benzene,
toluene,xylene
e
Nonhalogenated 2-methyl phenol
phenolics a n d
cresols
Polynuclear
aromatic
hydrocarbons
Alkanes
alkenes
Creosote, benzo-apyrene, anthracene
e
0
e
Trichlorophenol
Chlorophenols
Pentachlorophenol
Nitrogen
heterocyclics
Pyridine
Alkenes
e
a n d Fueloil
Polychlorinated
biphenyls
Chlorinated
solvents
Alkanes
Using
Anaerobic
Biodegradation
Process
e
0
0
e
e
Chloroform
e
e
Trichloroethylene
e
e
459
'
Some of the compounds listed above are thought by some to be extremely
bioresistant or not biodegradable at all. This may be the result of the inability
to treat some of these compounds in conventional wastewater treatment
reactors, which are not well suited to the treatment of many toxic o r
bioresistant compounds. Other reasons may include the use of the wrong
biological regimen (i.e. anaerobic, facultative, aerobic), or perhaps another
limiting factor was a t work in a particular attempt t o test the efficacy of
biological treatment. Many wastes and contaminated media contain
mixtures of chemicals, the combined effect of which is a resistance to
biological treatment. Some wastes have been shown t o be biodegradable only
under laboratory conditions (PCBs for example), and whether or not
biotechnology alone can effectively and economically degrade them has yet to
be shown.
The biodegradability of many chemical compounds can often be indicated by a
search of the scientific literature. Biotreatability investigations are needed to
determine if biotechnology is applicable for a given waste stream,
contaminated ground water or soil. The biodegradability of a compound or
mixture depends primarily on its chemical structure, concentration, toxicity,
and bioresistance. Far more compounds can be biodegraded than cannot.
OrganiCLoadingRate
It is as important to control organic loading rates in biological reactors used
to treat hazardous wastes and contaminated soil and ground water, as it is t o
control the same in conventional biological wastewater treatment plants.
Acceptable organic loading rates should be determined experimentally in
biotreatability investigations prior to system design. The organic loading rate
represents the mass of substrate presented t o the biological reactor to a
certain mass of microorganisms during a specified period of time. The
substrate represents food to the microorganisms, and the term Food-toMicroorganism Ratio (F/M) describes the rate of organic loading to a
biological reactor. In the design of wastewater treatment plants the food or
substrate concentration is usually measured by analyzing the waste for
BODS, COD or TOC. F/M can be computed by dividing the food value (BODS,
COD, TOC, or other value, in mg/l) by the concentration of biomass (MLVSS,
mg/l) multiplied by the hydraulic detention time, in days: F/M = BOD5, mg/l /
MLVSS, mg/l * DT, days.
On a BOD basis an F/M ratio range of 0.1-0.3 will normally provide the
necessary degree of treatment. Higher loading rates are possible for some
waste streams. Generally, F/M values greater than 0.3 result in partially
treated substrate which can result in increased toxicity to the microbes, and
a corresponding reduction in microbial diversity and population. F/M values
of less than 0.1 can produce acceptable contaminant removals, even with the
corresponding decline in microbial diversity and population. Basing F/M
calculations and reactor volume on a BOD basis, however, is most useful for
readily degradable wastes. For wastes that are bioresistant or inhibitory,
460
organic loading rates should be calculated using COD, TOC or other organic
loading indicator parameter and related t o chemical removal efficiency.
Once a method for measuring organic loading rate has been established, the
F/M ratio should be related to the respiration rate of the biomass. For aerobic
systems the Specific Oxygen Uptake Rate (SOUR)measurement is a reliable
measurement of oxygen utilization, or respiration. The test is conducted by
placing a sample of the biomass in a BOD bottle, inserting an oxygen probe
and plotting the consumption of oxygen over time, usually 15 minutes or less.
MLVSS is determined and respiration is calculated as a function of grams of
oxygen consumed grams of MLVSS per day. In aerobic systems a SOUR
range of 0.1 to 0.4 g 02 / g MLVSS-day generally indicates a healthy system.
SOUR rates of less than 0.1 g 02 / g MLVSS-day (but generally not less than
0.05 g 02 / g MLVSS-day) can indicate acceptable respiration for reactors
operated in the extended, or low F/M, mode. SOUR rates of less than 0.05g 02
f g MLVSS-day generally indicates inhibition o r loss of substrate.
For anaerobic systems the measurement of methane and CO2 gas production
can be used to measure respiration rate. Measuring the rate of respiration
and relating this rate to F/M and target compound residuals is important in
the design of the biological system. It is also important as an operational tool
to monitor system performance. This is particularly important for the
remediation of contaminated soils, as the nature and concentration of
contaminants i n soils can be expected t o change as their excavation
progresses.
For routinely generated hazardous wastes and for
groundwaters, their character and concentration may change somewhat or
gradually over time, and the frequent measurement of respiration rate is less
important.
Sudden changes in respiration rate is an indicator to the reactor operator
that a change in the system has occurred. A sudden decrease in respiration
rate may indicate a reduction in organic loading or the introduction of
inhibitory chemicals. A sudden increase in respiration rate signals an
increase in organic loading rate and alerts the reactor operator that control
of F/M is being lost. A sudden cessation in respiration indicates that the
biomass has been killed and a shutdown of the reactor is required until the
offending limiting factor or toxic condition has been addressed. Gradual
decreases in respiration rate with constant organic loading can indicate the
accumulation of toxic compounds in the biomass or the introduction of less
degradable substrate or inhibitory compounds. Gradual increases in
respiration rate with constant organic loading can indicate improved system
acclimation and biooxidation or the introduction of substrate which is easier
to degrade.
461
TOXIC CONCENTRATION LIMITING FACTORS
When evaluating biotechnology for a treatibility application, the
concentration of the substrate compounds is a very important factor. The
concentration of toxic chemicals can be viewed as a “dose” to a biological
system. The dose determines whether the chemicals present represent a
degradable substrate or a poison to the system. Sometimes the toxic
concentration limiting factor can be overcome by dilution of the bioreactor
feed, chemicaVphysica1 pretreatment of the waste, use of the appropriate
biological regimen or combinations of them, substrate augmentation, carbon
addition, bioaugmentation, or by addressing other degradation rate limiting
factors. Some of these corrective measures may be relatively simple in the
laboratory, but can be very difficult to implement in full scale operations.
Dation
Dilution, for example, is relatively easy for groundwater or light slurries. A
slurry tank preceeds the bioreactor, and effluent, wastewater, or makeup
water is proportioned into the feed tank t o reduce influent feed
concentrations. For treating contaminated soil or concentrated chemical
sludges on a continuous basis, dilution is more difficult. Making slurries
Erom soils requires experience in adapting solids handling, heavy equipment
and making the appropriate slurry dilutions. Pumpable slurries are easier
to dilute with the use of variable rate slurry pumps. Fortunately, most
biological treatment of concentrated wastes and contaminated soils will be
done on a batch basis, and the batch dilution of waste feedstocks is relatively
easy. Dilution of a feedstock for toxic chemical concentration reduction has
certain drawbacks. Increasing the volume of waste requires an increase in
the size of the bioreactor. This in turn, increases requirements for mixing,
aeration, temperature control and other environmental factors.
PhysicaVChemcalWaste Pretreatment
Waste pretreatment for addressing the toxicity concentration liming factor is
an option that may be useful in certain applications. Waste pretreatment
could be used t o partially degrade a compound, remove certain waste
components or reduce toxic chemical concentrations to manageable levels.
For contaminated ground water, pretreatment unit operations are in wide
use, especially carbon adsorption and air stripping units for ground water
treatment prior to discharge to publicly owned treatment works. For
concentrated organic liquid wastes and contaminated soil, pretreatment unit
operations are in limited use or are currently in the planning stages.
Conway and Ross 131 have investigated pollutant concentrations that may
make pretreatment advisable, and data from their studies is presented in the
following discussion. Table 2 summarizes their findings with regard t o
pretreatment requirements and pretreatment method for certain wastewater
462
11
conditions. This information was developed for activated sludge wastewater
treatment plants, and has direct application only for similar biological
reactors. Inhibition levels for other types of biological reactors may be higher
or lower, depending on the system design.
Excess sulfides can be air stripped or precipitated. For many biological
treatment systems sulfide concentrations in excess of 100 mg/l require
pretreatment. Anaerobic treatment systems can tolerate a maximum
soluble sulfide concentration of 200 mgfl [41. High phenol concentrations are
pretreated using solvent extraction, carbon adsorption or dilution. Phenol
concentrations of from 70 to 300 mgfl can be toxic t o biological treatment
systems, depending on the type of phenolic compound involved. Excess
ammonia is pretreated by dilution, ion exchange or air stripping. Ammonia
concentrations in excess of 16,000 mg/l can be toxic t o biological systems and
generally require pretreatment. Ammonia concentrations of much less than
16,000 mg/l can be toxic to certain biological treatment systems. Dissolved
solids or salts can inhibit biological activity by creating high osmotic pressure
outside the cell membrane, causing transport difficulties for nourishment
into the cell and the removal of wastes out of the cell. The limiting factor for
dissolved solids is 10,000 mg/l (as TDS). High dissolved solids concentrations
can be mitigated by dilution or pretreatment using ion exchange.
Heavy metals are absorbed by biological flocs and films and can accumulate
to toxic concentrations. The tolerance for metals in biological systems varies
widely, depending on the treatment system regimen, configuration, and
waste mix. Wastes which contain both sulfides and metals, for example,
often precipitate the metals before presentation to the biomass. Conway and
Ross suggest that for aerobic biological treatment systems, that the limiting
concentration for lead in influent wastewater 0.1 mgfl. Lead concentrations
must be kept at 0.1 mg/l or less to avert toxic effects in the biomass due to lead
accumulation. The limiting concentration for copper, nickel, and cyanide is
1.0 mgA; for hexavalent chromium and zinc it is 3.0 mgA; and for trivalent
chromium the limiting concentration is 10.0 mgfl.
463
TABU3 2
Concentration of Pollutants That Make Prebiological
or Primary Treatment Advisable*
Pollutant or
Limiting Concentration
System Condition
Kind of Treatment
Suspended solids
>50 to 125 mgfliter
Sedimentation, flotation, or
lagooning
Oil or grease
>35 to 50 mgfliter
Skimming tank or
separator
Toxic ions
Pb
Cu + Ni + Cn
Cr+6 +An
Cr+3
10,l mgAiter
21 mgAiter
13 mgkter
110 mgAiter
Precipitation or ion
exchange
PH
c6, >9
Neutralization
A1kalinity
0.5 lb alkalinity as
CaCOdb BOD removed
Neutralization for excessive
alkalinity
Acidity
Free mineral acidity
Neutralization
Organic load variation
>2:1 to 4:l
Equalization
Sulfides
>lo0 mg/liter
Precipitation or stripping
with recovery
Phenols
>70 to 300 mg/liter
Extraction, adsorption, or
internal dilution
Ammonia
>1.6 gAiter
Dilution, ion exchange, pH
adjustment, or stripping
Dissolved salts
>10 to 16 gfliter
Dilution or ion exchange
Temperature
13"t o 38°C in reactor
Cooling or steam addition
464
Other pretreatment technologies not yet fully developed may have potential
future applications. Pretreating contaminated soils is a significant
challenge to investigators today. Solvent addition t o a feedstock can render
certain wastes amenable t o biodegradation. Certain petroleum sludges, high
in oil and grease, can be rendered more degradable with the addition of
solvents. The solvents, themselves, are then subject t o biodegradation.
Wilson [SI is currently investigating solvent extraction as a method of
removing PCBs from contaminated soils, using hexane and sodium
dodecylsulfate as extraction solvents. This technique may have application
for sites contaminated with PCBs and other contaminants. Supercritical
extraction is another emerging technology that may have future application.
Chemical dechlorination of compounds with high chlorine concentrations
may also be useful. PCBs, for example, have been shown to be subject to
chemical dechlorination using sodium hydroxide, leaving the easily
degradable biphenyl for subsequent biological treatment. Dechlorination
with quicklime or other caustic agent may also be possible.
CarbonAddition
Powdered activated carbon (PAC) can be added to a biological reactor t o
reduce the toxic effect of the reactor influent and to retain contaminants in
the system to promote biodegradation. PAC adsorbs both toxic pollutants and
oxygen. Biomass attaches to the PAC particles to which it has available both
oxygen and the substrate for metabolism. PAC can also adsorb some toxic
metals, thus accumulating and concentrating them in the system. By
adsorbing toxic organic pollutants and being incorporated into the biomass,
the pollutants are physically retained in the biomass and are kept in contact
with the biomass longer. This can be an extremely important attribute €or
PAC systems because while motile microbial cells may be able to use their
motility to maintain contact with substrates, non-motile cells which cannot
are able to grow around and encapsulate contaminants in close contact with
them, facilitating their degradation.
BIODEGRADABlLlTY OF TOMC CHEMICALS
The term ‘toxic organic’ compound refers to both the chemical compounds
found on the EPA Emuent Guidelines Division List of Priority Pollutants and
to other chemicals not on the list which exert toxic effects t o biological
systems. Other lists of ‘toxic organic’ compounds have been developed by the
EPA Contract Laboratory Program for Superfund program work, lists of
compounds regulated under the RCRA and CERCLA programs and lists of
compounds developed by state regulatory agencies. For the purpose of this
discussion we will refer to a ‘toxic organic’ compound as one which exerts a
toxic effect on any biological system. The majority of the EPA listed priority
pollutants can be removed through convention biological treatment. Table 3
provides a comparison of the biodegradability of toxic pollutants as predicted
by an EPA study to the average results of priority pollutant removals from
five treatment plants [41.
465
TABLE 3
Comparison of Biodegradability of Toxic Organic
Pollutants as Predicted by EPA Study
To the Five Plant Study Results
Volatile Compound
Acrylonitrile
Benzene
Bromomethane
Bromodichloromethane
Carbon tetrachloride
Chlorobenzene
Chloroet hane
Chloroform
Dibromochloromethane
l,l,-Dichloroethane
1,2-Dichloroethane
1,l-Dichloroethene
t- 1,2-Dichloroethene
1,2-Dichloropropane
1,3-Dichloropropane
Ethylbenzene
Methylene chloride
1,1,2,2-Trichloroethane
Tetrachlorethene
l,l,l-Trichloroethane
1,1,2-Trichloroethane
Trichloroethene
Toluene
Vinyl chloride
EPA
Degradation,
100
100
48
67
100
100
NA
100
56
100
100
100
100
92
100
100
100
36
100
100
59
100
100
NA
466
%t
Five-Plant Study:
Removal, %$
99
100
100
89
100
w
91
99
100
7%
99
100
48
97
48
99
75
93
27§
3%
72§
40§
100
100
TABLE 3 (cont'd)
Comparison of Biodegradability of Toxic Organic
Pollutants as Predicted by EPA Study
To the Five Plant Study Results
Basemeutral Compound
Acenaphthene
Acenaphtylene
Anthracene
Benzo (a) anthracene
Benezo (b) fluoranthene
Benzo (a)pyrene
Bis (2-ethylhexy1)phthalate
Butylbenzylphthalate
Dibenzo (a, h) anthracene
Di-n-butylphthalate
1,3-Dichlorobenzene
1,2-Dichlorobenzene
Diethylphthalate
Dimethylphthalate
Dioctylphthalate
Fluoranthene
Fluorene
Isophorone
Naphthalene
Nitrobenzene
Pyrene
1,2,4-Trichlorobenzene
EPA
Degradation,
100
98
92
35
100
NA
95
100
NA
100
35
29
100
100
94
100
77
100
100
100
100
24
467
%t
Five-Plant Study:
Removal, %$.
TABLE 3 (cont'd)
Comparison of Biodegradability of Toxic Organic
Pollutants as Predicted by EPA Study
To the Five Plant Study Results
EPA:
Degradation, %t
Five-Plant Study:
Removal, %$
%Chlorophenol
100
41
Acid Compound
!2,4-Dichlorophynol
100
91
2.4-Dimethylphenol
100
100
2,4-Dinitrophenol
100
84
2-Nitrophenol
100
T7
Pentachlorophenol
100
36
Phenol
100
98
2,4,6-Trichlorophenol
100
45
t Includes volatilization losses at 25°C and initial concentration of 5 mgh.
4 Based on arithmetic means.
8 Influent concentration less than 40 ppb.
NA = not available.
468
Removal of organic compounds in activated sludge systems as well as other
biological reactors is through air stripping, adsorptiononto the biomass and
biodegradation. Only a small portion of the pollutant removal is attributed to
adsorption (generally less that 1%).Air stripping is an important removal
mechanism for volatile organic pollutants and some chemicals can be
completely removed by stripping alone. When air stripping is not acceptable
or applicable, many biological reactors can be adapted to minimize
volatilization losses.
Table 4 illustrates specific removal mechanisms and efficiencies of some
priority pollutants [61 for a conventional activated sludge system. Similar
removals can be expected for aerobic systems treating contaminated ground
water. Lower stripping efficiency and similar adsorption and biodegradation
efficiencies can be expected for the treatment of concentrated hazardous
wastes.
As stated earlier, a majority of the priority pollutants and other toxic
organics will biodegrade, although some will degrade much more slowly
than others. Appropriate biological treatment system design, directed
toward the removal of specific compounds, can provide the needed level of
treatment. Biological treatment systems, such as the bioslurry reactor, are
designed for the removal of a number of specific chemical compounds--not
for BOD, TSS and ammonia removal which typically governs the design of
wastewater treatment systems.
469
TABLE4
Specific Removal Efficiencies of Priority Pollutants
Compounds
PercentTreatment Achieved
Stripping Adsorption Biodegradation
Nitrogen Compounds
Acrylonitrile
Phenols
Phenol
2,4-DNP
2,4-DCP
99.9
PCP
Aromatics
1,2-DCB
1,3-DCB
Nitrobenzene
Benzene
Toluene
Ethylbenzene
Halogenated Hydrocarbons
Methylene Chloride
1,2-DCE
l,l,l-TCE
1,1,2,2-TCE
1,BDCP
TCE
Chloroform
Carbon Tetrachloride
0.58
21.7
2.0
5.1
5.2
8.0
99.5
100.0
93.5
99.9
a.1
19.0
33.0
99.9
99.3
95.2
97.3
78.2
-_-0.02
0.19
97.8
97.9
94.9
M.6
91.7
0.50
0.83
1.19
1.38
33.8
78.7
64.9
Oxygenated Compounds
Acrolein
99.9
Polynuclear Aromatics
Phenanthrene
Naphthalene
98.2
98.6
Phthalates
Bis (2-Ethylhexyl) Phthalates
76.9
Other
Ethyl Acetate
98.8
470
E"lMENTALLIMITING FACTORS
Microbes live and reproduce in a wide variety of environments. Bacteria
have been found in the deep ocean, underground, and in the polar ice caps.
Some require a highly specialized environment, like the sulfate reducing
bacteria which live in the vicinity of active vents along the ocean floor. Some
can live and function well in almost any environment that can offer only
minimal support for life. The microbes that are most useful to the
environmental scientist or engineer, however, require that certain
environmental factors and sources of nutrition me carefully maintained, if
they are to function as we wish. The careful maintenance of environmental
limiting factors is vital t o maintaining the optimal rate of pollutant
degradation.
Ti?mp€WtltUt-e
Temperature is a very important environmental limiting factor.
Temperature affects the rate of cellular metabolism and the solubility of
substrate. An individual microbial species has an optimum and maximum
temperature for growth. At high temperatures extracellular enzymes can be
denatured, or cell death may occur. A t low temperature metabolic activity
decreases significantly. Psychrophiles microbes grow best at temperatures
below Z O O C. Mesophiles do best at 15" C to about 45" C. Most microbes are
grouped into this category. Thermophiles grow at temperatures above 45°C.
The microbes of interest to us are the Mesophiles. Most biological reactors
are operated at about 20" C. Since metabolic reaction rate can double for each
10" C increase in temperature, there can be some advantage to operating a
biological reactor at temperatures higher than those permitted by ambient
conditions. Temperature should be maintained at no less than 10" C and no
more than 35" C to 40" C. The cost of pumping calories into a reactor t o raise
temperature can be prohibitive unless a cheap source of steam or other heat
source is availible at the site. Heat in biological reactors comes primarily
from influent feed temperature, mechanical energy input, solar energy and
metabolic activity. Heat losses result primarily from aeration and heat loss
through reactor walls. For relatively small reactors the aeration system can
be designed to minimize heat loss, and the reactor can be insulated. For
large lagoon-sized bioremediation projects, it is rarely possible t o control
reactor temperature, except to cool the biomass by spray cooling or aeration.
For in-situ applications temperature cannot be controlled, and below ground
temperatures are quite low. This means that in-situ bioremediation will
proceed at relatively slow rates and will require longer time frames before
site remediation can be completed.
471
Most of the microbes of use in bioremediation applications are obligate
aerobes which use aerobic respiration t o obtain energy. For in-situ
bioremediation projects oxygen can’ be supplied t o the subsurface
environment by aerating recirculation water, or by injecting solutions of
hydrogen peroxide or ozone. For surface or marine spills atmospheric
oxygen is availible and supplemental sources are not needed.
The use of strictly anaerobic regimens t o treat hazardous wastes,
contaminated soils or groundwater will be limited to a few specialized,
substrate-specific applications. Anaerobic degradation of pollutants occurs
but the rate of degradation is much slower than that of aerobic degradation.
Facultative anaerobes can exist in environments in which anaerobidaerobic
conditions alternate, by sequentially substituting oxygen with nitrate as the
chemical electron acceptors. It is becoming apparent that for some
applications (like those for the degradation of chlorinated aliphatic
compounds) a combination of aerobic and facultative biological regimens is
needed, and precise oxygen control is essential. Aerobic/facultative biological
treatment systems can be designed by separating the reactor into separate
cells for sequential facultative o r aerobic treatment. The aerobidfacultative
regimen can also be maintained in a single reactor in which soil particles or
carbon are added as growth media and dissolved oxygen levels are kept low
(about 0.5 mg/l). Biological floc grows around the media particle and the
microbes attached to or nearest to the particle become covered by subsequent
growth. This outer growth is exposed t o the oxygen in solution while the
inner growth has limited exposure to oxygen. By keeping the dissolved
oxygen content low, the driving force of the oxygen into the floc is reduced
significantly, compared to saturated conditions. For the treatment of liquid
wastes or contaminated groundwater, recirculating biological contactors,
trickling filters, suspended growth biological filters, and other biological
systems provide the aerobidfacultative interface very efficiently.
Most bacteria (neutrophiles), protozoans and other microbes grow best at a
neutral pH. Acid producing bacteria (acidophiles) are adapted t o
environments at lower pH value. Some bacteria (basophiles) are adapted to
alkaline environments. Fungi can be grown in environments of extreme pH
better than most bacteria. Biological reactor pH is maintained in the 6-8 pH
value range by adjusting reactor feed pH. The optimum pH for biological
reactors is believed to be in the middle of this range Anaerobic biological
reactors often go “sour” when acid producing bacteria gain predominance,
resulting in biomass pH values of 5 or less. Careful monitoring of pH is
needed for these reactors, and the addition of caustic neutralizing agents and
sodium bicarbonate can help maintain pH control. For biological treatment
of surface or marine spills of petroleum products or other organic chemicals,
pH control in surficial soils is usually not required except in salt marshes
472
and other places where acid soils are present. For in-situ treatment
applications involving highly acid or alkaline ground water, biological
treatment may not be a viable option.
Nutrients
An inadequate supply of nutrients can severely limit the growth and
metabolism of microorganisms, thus limiting the rate of substrate removal.
Nitrogen and phosphorus are recognized as being needed in the greatest
quantity for cell synthesis and metabolism. Some industrial wastewater
treatment plants add phosphoric acid and liquid ammonia t o provide these
essential nutrients. In addition to nitrogen and phosphorus other nutrients
are needed by microbes. These include sulfur, magnesium, potassium,
calcium, and metallic elements including gold, boron, iron and other metals
in trace quantities. Fertilizer mixtures can be added to biological reactors to
provide needed nutrients. Nitrogen, phosphorus and other nutrients are
injected to the subsurface for in-situ bioremediation applications. Nutrient
addition in activated sludge treatment plants is provided at a B0D:N:P ratio
of 100:5:1. For landfarming operations a C:N:P ratio of 100:2:0.2 has been
found to be successful.
Essential micronutrients such as calcium, iron, metals and others are often
assumed to be present and available in the subsurface environment. This
may be the case in many locales, but should not be assumed to be the case
everywhere. It would be safer to assume that micronutrients are not present
a t a given site, and add them to promote optimum biological growth. For
biological treatment of surface or marine spills of petroleum products or
other organic chemicals, nutrients are applied to the spill site to stimulate
biodegradation of the pollutants. Approximately 110 miles of beaches in
Prince William Sound, Alaska were treated with nitrogen- and phosphorusrich fertilizers to accelerate the biodegradation of crude oil released to the
environment as a result of the Exxon Vuldez oil spill of March 1989.
Researches report that the rate of biodegradation was acellerated by 200-400%
as a result of nutrient addition 173.
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BIOLOGICAL CONSORTIA LIMITING FACTORS
The success of a given bioremediation application depends on the factors
mentioned previously and the development, acclimation and growth of a
biological regimen capable of degrading the target substrates. The factors
discussed below relate to the biomass itself; it’s regimen, diversity, consortia
development,and acclimation.
BiologicalRegimen
Biological regimens for biotreatibility applications are divided into anaerobic,
facultative, and aerobic systems. Aerobic systems are able to effectively treat
many waste mixtures but are subject to toxic concentration effects for others.
For many biotechnology applications it is becoming evident that anaerobic or
facultative systems, or combinations of them with aerobic systems, can be
used to degrade toxic or bioresistant compounds. In the wastewater
treatment field, combinations of biological regimens are widely employed t o
enhance contaminant removal rates in sequential biologocal reactors in
which different biological regimens are maintained. Use of the appropriate
biological regimen or combination of regimens is crucial to successful
biotechnology applications.
U d b
Microorganisms have evolved their capability t o degrade natural organic
compounds over a period of millions of years. Biodegradable man-made
compounds, which began to appear in the environment within the last 100
years, also provide a rich source of organic carbon and energy needed for
growth. The activity and growth of microorganisms in a biological reactor or
in the environment is largely governed by their ability to produce enzymes to
catalyze metabolic reactions. Lack of an appropriate enzyme-or the species
of microbe that produces it-can mean a failure to break down a parent
compound or the inability t o completely degrade daughter compounds.
Hydrolytic enzymes are secreted by the cells into the biological medium.
These enzymes break higher molecular weight compounds into lower
molecular weight compounds. Some compounds and ions can pass through
the cytoplasmic membrane into the cell by free diffusion, which is dependent
on the concentration gradient of the compound between the inside and
outside of the cell membrane. Certain proteins exist inside the cytoplasmic
membrane which couple substrate transport to an energy-yielding process.
This mechanism, called active transport, is the principal means of cell entry
for most substrates and ions. These proteins can be very specific for a
particular substrate, even t o the exclusion of very similar or chemically
related substrates. A biological consortia with a high diversity provides a
biological system with a larger variety and concentration of enzymes and
metabolic systems capable of metabolizing a larger number of substrates.
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Biological diversity, therefore, is an important factor in developing the
biological consortia to be employed for a given application. Addressing
potential limiting factors thoroughly ensures that the maximum system
diversity possible is available.
Startingthe Biological Consortia
.
Municipal sewage treatment plant mixed liquor or waste activated sludge is
most often used t o seed biological reactors. This source of microorganisms is
often sufficient for starting aerobic or facultative reactors. Anaerobic
digester sludge is used as seed for anaerobic reactors and to supplement
facultative systems. The optimum biological consortia for a given project will
almost always consist of a population of microbes with moderate t o high
diversity. Municipal wastewater treatment plant biomass (especially
biomass treating industrial wastewaters as well as sewage) provides a good
basis for culturing a biological consortia. For most applications municipal
treatment plant biomass will be suficient. For some applications, however,
the biological consortia needs to be supplemented with additional substrate,
nutrients or microorganisms for optimum substrate degradation. Other
sources of microorganisms include contaminated soil, horse manure, and
cultured microorganisms.
Most surficial organic soils contain up to 1x 106 microbial cells per gram of
soil. Some contaminated soils will contain microbes already adapted to the
degradation of a contaminant or mixture of contaminants. These can be
excavated and cultured into a biological consortia which is already adapted
and acclimated for the degradation of the target compounds.
There is increasing interest in providing specific groups of microbes-those
best suited t o provide the needed degree of treatment-for a specific treatment
application. Highly specialized biological consortia have been cultured and
produced on a commercial scale for the treatment of specific classes of
chemicals or wastes. Microbial cultures have been developed for the
treatment of phenolic and cyanide compounds, aromatic hydrocarbons,
petroleum products and spills, for the nitrification of ammonia t o nitrate,
and for other specific purposes. These cultures are available in powder,
freeze dried and liquid forms. Bioaugmentation, the use of supplemental
cultured microorganisms, may be useful in some applications. One problem
with bioaugmentation is that in many cases the biological supplement must
be continuously added. The bacteria generally work well in degrading a
given substance, but the beneficial trait that gives the strain(s) an advantage
in degrading a particular compound sometimes disappears after the
mutation of a number of generations.
Biological supplements are in wide use for the breakdown of oil and grease in
a wide variety of applications ranging from the degreasing of sewage
pumping station wet wells to the cleaning of bilge tanks of ocean going
vessels. Biological supplements can sometimes prove superior to naturally
475
ocurring organisms, but not always.
Researchers investigating
bioremediation alternatives during the Exxon Vuldez oil spill cleanup,
however, were unable to demonstrate any advantages t o bioaugmentation for
the products tested [7].
Acclimation
After seeding a biological system a period of acclimation is needed t o
condition the microbes to the substrate. The microbes need time to adapt to
the substrate and develop the metabolic pathways needed for them to exploit
substrate carbon and energy. Some compounds can require several weeks
for acclimation to occur. Benzidine, for example, has been shown to require
up to six weeks for acclimation to occur. Figure 1 represents an acclimation
curve for benzidine for an activated sludge application.
Acclimation can be accomplished on either a batch or continuous basis.
Waste is added gradually to a biological reactor at an organic loading rate a t
an F/M of less than 0.1 (BOD basis) for readily to moderately biodegradable
compounds. For bioresistant or toxic compounds, batch laboratory studies
can be used to develop acclimation feed rates. It is important not to shock the
microorganisms during the acclimation process as i t would then possibly be
necessary to reseed the system and start over again. Acclimation can be
observed by examining bacterial growth by measuring MLVSS
concentrations, using culture and counting methods, analyzing the biomass
for adenosine triphosphate concentrations, by measuring biomass
respiration rate, and by measuring emuent or filtered mixed liquor target
compound concentrations.
476
10
I
I
I
I
8
6
4
2
I
I
I
I
2
0
WEEKS OF ACCLIMATION
FIGURE
1
ACCLIMATION FOR THE DEGRADATION OF BENZIDINE
477
C
~
Time, Hours
IC
Figure 2. Bacterial Growth Curve. A, lag or acclimation phase;
B, logarithmic growth phase; C, stationary phase; D, declining
phase (endogenous phase); E, surviving population.
The bacterial growth curve (Figure 2) illustrates bacterial growth in a batch
system. The biological system starts out with a nominal seeded population.
Substrate is introduced and initially there is no change in population. This is
the lag or acclimation phase. Upon acclimation to the substrate, the bacteria
enter a period of unrestricted multiplication called the log or exponential
growth phase. Under optimal conditions a bacterial cell can divide every 15
minutes. The increase in microbial numbers can be observed by using the
methods described above. When a nutrient or carbon source becomes a
limiting factor, or when toxic or inhibitory compounds accumulate, the
biomass enters a stationary phase characterized by no net growth. The
bacteria then enter a stage of endogenous respiration and declining
population and diversity. With no new carbon and energy available, the cells
metabolize their o w n protoplasm until death occurs. When death occurs the
cells lyse, releasing nutrients and food to the system which is consumed by
other cells. In this way a surviving population of microorganisms can exist
for some time. In continuous feed systems the population will increase until
substrate concentration or another limiting factor governs the rate of growth
of new cells. The rate of cell death corresponds to the rate of cell growth.
478
For both batch and continuous feed systems it is important to monitor the
biomass t o determine the health, activity and growth phase of the system.
Having successfully acclimated the system, feed rate should be increased t o
the point of maximum cell growth rate, without overdosing or shocking the
system.
During acclimation of a system intended to treat toxic o r bioresistant
compounds, it is often the practice to supplement the target compound with a
more degradable food source, enzymes, folic acid or other supplement. This
is done to provide optimal support for the microbes while they mutate strains
that can exploit the toxic or bioresistant substrate. Also during acclimation,
it is a common practice t o provide a surplus of nutrients t o the system. When
the microbes enter the maximum growth phase and the system stabilizes,
nutrient addition is scaled back.
Biomass acclimation is an important phase in the development of a biological
system. All of the limiting factors discussed previously should be addressed
during acclimation t o ensure the optimum growth and operation of the
system.
SubstrateAugmentation
Substrate augmentation involves the addition of supplemental organic
substrate t o promote the viability and growth of biomass in order to degrade
target organic compounds. Substrate augmentation, in some cases, will
enhance the rate of biodegradation of target contaminants. Substrate
augmentation chemicals are added to bioreactors t o provide carbon t o a
system in which carbon as a source of energy is a limiting factor and/or, t o
stimulate the microbial production of enzymes or other metabolites needed t o
degrade a particular contaminant.
Many contaminated aquifers do not contain sufficient readily degradable
carbon to support an active biomass, and for these applications substrate
augmentation is necessary. With supplemental carbon the biomass can
grow to concentrations and diversity with sufficient energy t o biodegrade the
target contaminants.
Wilson and Wilson 181 have experimented with the addition of methane to an
unsaturated soil column in the laboratory to stimulate the production of
monooxygenase to promote the degradation of TCE. They showed that
methane oxidizing bacteria (methanotrophs) can degrade TCE via the
phenomena known a s cometabolism.
Methanotrophs excrete
monooxygenase, a metabolic enzyme, for the initial step in the oxidation of
methane, which the organisms use for energy and growth. Monooxygenase
also is able t o oxidize other hydrocarbons, and appears t o cause the
epoxidation of chlorinated alkenes [91 such as TCE, TCA and others.
Chlorinated alkene epoxides hydrolyze in water t o a number of compounds
which are quite readily mineralized by other bacteria.
479
McCarty, et. al., proposed a biological treatment system combining in-situ
and above ground biological systems which feature the injection of methane
and oxygen t o the subsurface environment t o initiate and suppport
cometabolism and mineralization of chlorinated aliphatic compounds.
Adding methane in this manner consitutes substrate enhancement, and
illustrates the potential usefulness of this substrate enhancement technique.
480
References:
1.
U.S. Environmental Proctection Agency, Bioremediation in the
Field, EPA/540/2-91/007, No. 2, March 1991.
2.
U S . Environmental Proctection Agency,
Bioremediation, EPA/540/2-9 1/002
3.
Conway, R.A, and R.D. Ross. 1980. Handbook of Industrial Waste
Disposal Van Nostrand Reinhold Co., New York.
4.
W. Wesley Eckenfelder, Industrial Water Pollution Control, 2nd
ed., McGraw-Hill Book Company
5.
Wilson, David J., Soil Clean Up by in-situ Surfactant Flushing,
Separation Science and Technology, 23(11), pp.863-892,1989.
6
Kincannon, D. F. and Stover, E.L., Determination of Activated
Sludge Biokinetic Constants for Chemical and Plastic Wastewaters,
EPA DraR Report, CR-806843-01-02,1982
7.
U.S. Congress, Office of Technology Assessment, Bioremediation
for
Marine Oil Spills-Background Paper, OTA-BP-0-70
(Washington, DC:U.S. Government Printing Office, May 1991).
8.
Wilson, John T. and Wilson, BArbara, H., Biotransformation of
Trichloroethylene in Soil, Applied and Environmental Microbiology,
Jan. 1985, pp.242-243.
9.
McCarty, P.L., Semprini, L. and Roberts, P.V., Methodologies For
Evaluating The Feasibility On In-Situ Biodegradation Of
Halogenated Aliphatic Groundwater Contaminants By
Methanotrophs.
481
Understanding
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