x>EPA
United States
Environmental Protection
Agency
Office of Emergency and
Remedial Response
Washington DC 20460
Superfund
EPA, 540/2-90/012
Office of Research and
Development
Cincinnati OH 45268
September 1990
International Evaluation of
In-Situ Biorestoration of
Contaminated Soil and
Groundwater
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EPA/540/2-90/012
September 1990
INTERNATIONAL EVALUATION OF IN-SITU BIORESTORATION
OF CONTAMINATED SOIL AND GROUNDWATER
Sjef J.J.M. Staps
formerly of:
National Institute of Public Health
and Environmental Protection
P.O. Box 1
3720 BA Bilthoven
The Netherlands
current contact:
Grontmij N.V.
P.O. Box 203
3730 AE DeBilt
The Netherlands
Telephone: 31-30-20-79-11
reprinted from:
Proceedings of NATO/CCMS Third International Conference
Demonstration of Remedial Action Technologies
for Contaminated Land and Groundwater
Montreal, Canada
November 6-9, 1989
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FOREWORD
The Environmental Protection Agency is a participant in the
NATO/CCMS (Committee for the Challenges to Modern Society) Pilot
Study on Demonstration of Remedial Action Technologies for
Contaminated Land and Groundwater. The purpose of this project is
to develop and share information on new and innovative technologies
for remedial action at hazardous waste sites. Over 400 scientists
from 13 countries have been involved in this project to date. This
cooperative effort is in response to a common need among
industrialized countries for technologies which can provide more
cost-effective means for site remediation. This paper resulted
from work under a NATO/CCMS fellowship and will be published in
1992 as part of a comprehensive report for the entire NATO/CCMS
study. In the meantime, we decided to publish it separately due
to the strong current interest in the potential use of bioremedia-
tion for hazardous waste cleanup. This paper provides a timely
overview of the status of in-situ bioremediation technology and
should be of interest to those working on problems at Superfund,
RCRA, and underground storage tank sites.
Walter W. Kovalick, Jr., Ph.D.
Director,
Technology Innovation Office
Donald E. Banning
Director,
NATO/CCMS Pilot Study
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ABSTRACT
This-paper is the result of the RIVM-project "International evaluation of in-
situ biorestoration of contaminated soil and groundwater". As a fellowship
project, it was associated with the international NATO/CCMS pilot project on
"Demonstration of Remedial Action Technologies for Contaminated Land and
Groundwater".
The philosophy of in-situ biorestoration is to stimulate the indigenous soil
microorganisms to degrade contaminants by improving the environmental
conditions in the soil using a water recirculation system.
The objective of the project is to show the possibilities for application of
the technique in relation with contaminants, soil conditions and other site-
specific circumstances by means of integration and evaluation of results of
in-situ biorestoration projects.
The project is limited to the Netherlands, West Germany and the USA. It was
implemented by visiting 23 relevant projects in these three countries, which
play a leading role in the development of remediation techniques for
contaminated soil and groundwater.
In-situ biorestoration is a relatively young, developing technology. It has
been used at several locations, mainly in the USA. It can be used especially
for locations at which both the unsaturated zone and the groundwater are
contaminated with hydrocarbons. A precondition is a good permeability of the
soil.
Experience has especially been gained with in-situ biorestoration at
hydrocarbon-contaminated petrol stations and industrial sites. The system
generally consists of a water recirculation system, aboveground water
treatment and conditioning of the infiltrating water with nutrients and an
oxygen source. However, there is no one-and-only application method for in-
situ biorestoration. The remediation, which can last from approximately six
months to several years, can reach residual concentrations below the B-value
of the Netherlands examination framework (see table 4). If applicable, in-situ
biorestoration is generally more cost-effective than other remediation
techniques; costs are approximately between 40-80 US $/m .
Recommendations from this evaluation include a further stimulation of the
development of this technology, improvement of the preliminary research,
expansion of the applicability to more recalcitrant contaminants, research on
bio-availability and research into oxygen .supply and distribution in the
subsoil.
INTRODUCTION.
In behalf of the Dutch clean-up operation for contaminated soil, development
of adequate clean-up methods is considered to be of prime importance. Besides
thermal and extraction techniques, which still account for the greater part of
the clean-up operation, biological techniques have been developed in the
Netherlands. Landfarming, a biological treatment technology for excavated
contaminated soil, is now being used on a practical scale (Socz6 and Staps,
1988). However, in many cases it is impossible or too expensive to excavate
1 in (its original) place
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the soil. In-situ techniques are then the most appropriate methods, and can be
employed for treating both the soil and the groundwater.
In the Netherlands, application of in-situ techniques by companies nowadays
focusses on washing, circulating and cleaning of the groundwater (the former
pump-and-treat method). Especially in recent years, increasing interest is
also being shown in actual biorestoration in the soil. The environmental
conditions in the subsoil are optimized by supplying oxygen and nutrients and
circulating the water.
The first Dutch research into in-situ biorestoration was a feasibility study,
carried out by Delft Geotechnics, to evaluate the scope of an in-situ soil-
venting technique (van Eyk and Vreeken, 1988). The RIVM and the TNO
(Netherlands Organization for Applied Scientific Research) are preparing a
full-scale clean-up by means of a literature study and extensive experiments
on laboratory-scale since 1985 (Verheul et.al., 1988). However, a clear need
for information from full-scale clean-up projects and from foreign experience
was still felt. From literature and international contacts is was known, that
especially in the USA experience with this technology had been gained.
Besides, developments were also under way in West Germany (Nagel et.al., 1982
and others).
While the problem of soil and water contamination also became evident in other
countries, interest in remediation techniques, and especially in-situ
technologies, increased. This emerged at the first international workshop of
the NATO/CCMS pilot project on "Demonstration of Remedial Action Technologies
for Contaminated Land and Groundwater" in spring 1987. Several western
countries, including the Netherlands and the USA are participating in this
pilot project.
This was sufficient reason for the RIVM to start this evaluation in late 1987.
The author was awarded a fellowship of the NATO/CCMS project. Because of its
relevance to the development of remediation techniques in the Netherlands, the
study is partly financed by The Netherlands Integrated Soil Research program.
The project is limited to the Netherlands, West Germany and the USA. The
fellowship project was implemented by visiting 23 projects in this field in
these three countries, which play a leading role in the development of
remediation techniques for contaminated soil and groundwater. An overview of
the projects is given in the appendix.
Information, results and data are directly obtained from the experts involved.
Total information is arranged,.and conclusions are drawn in this final paper.
A more comprehensive report, including detailed information from the visited
projects, is in print (Staps, 1989 ). Information concerning analytical
procedures is also included in this report.
EVALUATION OF IN-SITU BIORESTORATION PROJECTS
Introduction
Although not all organizations dealing with in-situ biorestoration are
included, the 23 projects chosen do provide a good idea of the feasibility of
this technology. The concerning group of 23 organizations consisted of fifteen
private companies, three institutes, two universities, one co-operation of an
institute, a university and a coast guard, and one air force.
A schematical overview of the projects, including several characteristics, is
given in the appendix. Projects U8 and U9 cannot really be regarded as in-situ
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blorestoration projects, because in both cases biprestoration does not take
place in the original location. The clean-up site in project U8 is a lagoon,
and in the case of U9, the clean-up consists of on-site landfarming. These two
projects are not included in this chapter, but, because of the direct
relationship to the other projects, they are included in the general overview.
Other divergent projects are N5 and U5; both are research projects, where the
contamination has been caused deliberately. Moreover, project N5 is deviating
because the biostimulation is performed only by venting of the soil, and thus
is limited to the unsaturated zone.
Projects D5 and D6 differ from the other ones in that they are
conceptual phase, and data from demonstration scale
available.
A substantial proportion of the remaining group
biorestoration projects is characterized by research
still in the
test are as yet not
of "real" in-situ
aspects, with the
majority having been set-up as a research project (Nl, D2, U3, U5, U6).
Background of the sites at which in-situ biorestoration has been or is
applied
being
The locations at which in-situ biorestoration has been or is being applied can
be divided into two main groups:
- filling stations (service stations, airforce bases, marshaling yards, bus
stations) with leaking pipelines or storage tanks,
- chemical industry sites, mainly (former) refineries.
All locations were contaminated with hydrocarbons, for the most part defined
as petrol and/or diesel. At airforce bases, also kerosene or JP-4
contaminations occur. One-fifth of the projects concerned chlorinated
hydrocarbons. The smallest group of locations was contaminated with PAHs or a
mixture of chlorinated hydrocarbons, mineral oil and PAHs. The latter has not
yet been demonstrated.
Preliminary site characterization
The surface area of the sites at which2 in-situ biorestoration was applied,
varies largely; from 20 to 75,000 m .Within this variety, two clear groups
can be distinguished. The first group is2 formed by filling stations; the
surface area is mainly 400 - 1,000 m . The second group consists of large
chemical industry and (former) yefinery sites, and here, the area is varying
between 20,000 and 75,000 m . The depth upto which the contamination is
dispersed is generally between 3 and 10 meters below surface level.
It was striking that the discovery of a second contamination during the
cleanup-process occurred at several projects.
In relation with soil structure and geology, nearly all locations can be
defined as sandy. At several places, clay layers are present. Only in an
exceptional case, in-situ biorestoration is applied at a site with overburden
clay and fractured bedrock.
Concerning geology, permeability is a very important parameter for in-situ
biorestojation. For the projects3reviewed, the Kf-value varied between 10
and 10" m/s, mainly having 10" -10" as order of magnitude. Generally, a Kf-
value of 10" is regarded as being the minimum permeability for successful
application of in-situ biorestoration.
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Preliminary biodegradation research
In order to decide whether in-situ. biorestoration can be applied at a
contaminated site, microbiological, hydrogeological and chemical aspects must
be regarded. Hydrogeological conditions include permeability, dispersion of
contaminants, groundwater level and flow. The parameters which might be
considered before chosing or designing in-situ biorestoration are:
- Microbial parameters (total cell count, nitrifiers, denitrifiers,
. hydrocarbon degraders)
- Oxygen demand
- Nutrient demand
- Contaminant degradation rate
- Bio-availability.
Total cell count forms the base for research on populations of microorganisms.
Parameters in relation with biological activity are an important part of
microbial research. For in-situ biorestoration, the number of metabolic active
organisms and enzyme samples are important as an indicator for biodegradation
in the subsurface. As regards hydrocarbon contamination, determination of the
percentage of hydrocarbon degraders is an important monitoring aspect too.
Besides, there is a large group of relevant physical and chemical parameters,
including permeability, pH, oxygen, redox conditions, temperature, TOG, DOC,
BOD, Fe-concentration, Mn-concentration, concentration of (heavy) metals,
N , , ammonium-concentration, nitrate-concentration, nitrite-concentration
ana pnosphate-concentration.
A high permeability is one of the conditions for a successful in-situ
biorestoration.
Soil pH may affect sorption of ionizable compounds in addition to limiting the
types of microorganisms in the subsurface. Hethanogenes, which have been
implicated in mineralization of some aromatic hydrocarbons, are inhibited at
pH values below 6 (Lee et.al., 1988).
Biodegradation of many organic pollutants in the subsurface may be limited by
insufficient concentrations of oxygen or unfavourable redox conditions.
Also temperature influences microbial metabolism of subsurface pollutants. The
temperature of the upper 10 m of the subsurface may vary seasonably. However,
in the Netherlands, it will not deviate-much from 10°C. Also below 10 m,
temperature will be about this value. It is important to keep this in mind
when comparing results from projects in for example Florida (U6) or California
(U5) where much higher temperatures (20-25 C) are measured with projects from
other regions.
Total organic carbon (TOO, dissolved organic carbon (DOC), chemical oxygen
demand (COD) and biological oxygen demand (BOD) are sum parameters. TOC and
DOC are direct parameters for the carbon concentration of organic compounds.
Decreasing concentrations of TOC and BOD values indicate mineralization of the
organic contaminants.
Determination of Fe and Mn concentrations is important because high
concentrations of these metals can cause precipitation under aerobic
conditions, caused by the infiltration of oxygen during the biorestoration
process.
Other heavy metals can be important, especially at contaminated sites, because
at toxic levels, they can inhibit the activity of microorganisms.
Inorganic nutrients like nitrogen and phosphorus may be limiting when the
carbon/nitrogen/phosphorus (C:N:P) ratio is unfavourable. Determination of
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ammonium, nitrate and nitrite gives insight in the stage of the conditions in
the subsoil.
After the characterization of the site regarding microbiological,
hydrogeological and chemical/physical parameters, a first decision can be
taken whether in-situ biorestoration is applicable at a specific site.
However, there is no one-and-only application method for in-situ
biorestoration. If the option of in-situ biorestoration is chosen, nearly all
visited organizations perform preliminary biotreatment studies on laboratory
scale to get insight in the optimal stimulatory actions for a biodegradation
process at the site and to choose the right combination of microbial,
hydrogeological and physical/chemical actions. Only organizations with very
broad experience in the field of pump-and-treat and in-situ biorestoration
design a site-specific in-situ biorestoration system almost directly based on
the site characteristics (Ul, U7). A large majority of the projects included
preliminary • laboratory research, both small-scale tests and percolation
studies in columns. In a few cases, field experiments in a small area
representative of the contaminated site have also been performed.
System design
Description of the installation
In-situ biorestoration involves the stimulation of the biodegradation of
contaminants at contaminated sites without excavation of the soil. In this
process, the soil of the contaminated location is used as a bioreactor (see
figure 1). ,,; ,
The specifications of the "bioreactor11 in the subsoil are based on the
characteristics of the contaminated site, and the objectives and requirements
of the clean-up. They include for example the type and distribution of the
contaminants in the subsoil, the soil geology and hydrology and the need for
isolation of the location.
In most cases a semi-closed configuration is used in such a way, that the
contaminated location is isolated and controlled; uncontaminated groundwater
can enter the contaminated site, but contaminated groundwater cannot move to
uncontaminated areas.
The site can be isolated using hydrological intervention technologies or civil
engineering operations. In general, a hydrological system is. designed, in
which the groundwater is centrally withdrawn, and after above-ground
treatment, reinfiltrated at several points on the periphery of the location.
The groundwater is withdrawn at a higher rate than it is infiltrated, the
surplus generally being discharged into a sewer.
To support degradation in the subsurface, an above-ground treatment system is
used to degrade the contaminants in -the withdrawn groundwater, and to
condition the water before re-infiltration.
Biodegradation relies entirely on the contact between the contaminants (in the
water phase) and the microorganisms. In the case of highly volatile compounds
as contaminants, clean-up can partly be achieved by vaporization of the
unsaturated zone using a soil venting system, as is shown in project N4. The
contaminated exhaust air can be treated above ground by adsorbtion (e.g.
activated carbon) or oxidation in a biological, thermal or catalytic manner.
Research project N5 describes the design of an in-situ soil venting system,
used both as a physical (evaporation) and a biological process
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(biodegradations). This system can be used for contaminations in the
unsaturated zone.
compost filter
catalytic oxidation
Addition of H202/N°3/02/Qir/°3
Addition of nutrients
Addition of micros-organisms
Heating '
discharge /•>
well
- land level
contamination
groundwater-level
^3 discharge well
.
11 monitoring well
11 monitoring well
clay layer
Figure 1. General scheme of in-situ biorestoration.
There are several options for reinfiltration:
- injection wells
- infiltration galleries
- surface application.
Infiltration into the saturated zone through injection wells is the most
direct method, but also has disadvantages: oxygen and nutrients are only
poorly delivered to the unsaturated zone and the wells have small surface
areas. Therefore, they can prone to clogging. The installation cost decreases
in the order: injection wells, infiltration galleries and surface application.
When visiting the projects, it appeared that infiltration galleries were used
nearly twice as often as injection wells. Surface application is only rarely
used.
At one research project (U6, Downey 1988), the three options were used
simultaneously in order to gain insight in their applicability.
As regards the above-ground treatment, the first part is generally a sandbox.
Undissolved contaminants are removed in an oil/water separator. An air
stripper is used to remove volatile contaminants. In a few projects,
biological systems, such as a trickling, filter, were used for degrading
dissolved compounds.
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When required by the legislator, the contaminated air from the air stripper is
oxidized in order to bring about degradation of the contamination instead of
moving the contaminants from one compartment (groundwater) to another (air).
In the Netherlands, this is performed using a biological compost filter, in
which adapted microorganisms degrade the contaminants. In the USA, catalytic
oxidation systems are employed.
Hydrological aspects
In general, in-situ biorestoration is performed by means of saturating the
subsoil. The main hydrological steps taken consist of central withdrawal of
the groundwater and reinfiltration at several injection points on the
periphery of the location. Groundwater is withdrawn at a higher rate than it
is infiltrated. This occurred at about 95% of the locations.
At two projects, in-situ biorestoration was performed without water saturation
(D2 and D4). However, saturating the soil makes it easier to optimize the
environmental conditions in the soil with respect to other parameters like pH,
oxygen content, nutrients, etc. It depends on the site-specific situation
whether saturation and other optimizations will be chosen, or no saturation
and fewer other optimizations. However, in most cases, saturation is the
preferred method.
Oxygen supply
As far as is known, in-situ biorestoration has only been applied to
hydrocarbon-contaminated sites. In order to initiate hydrocarbon oxidation,
microbial populations utilize oxygen:
C6H6 4- 7k02 -* 6C02 + 3H20 (for benzene) .
As a result of the contamination, the subsoil of contaminated sites is
anaerobic, or contains very low concentrations of oxygen. Therefore, oxygen
has to be supplied for in-situ biorestoration. Sources of oxygen include air,
pure oxygen and hydrogen peroxide. Subsequent oxidation can also be sustained
by alternative electron acceptors, for example nitrate.
Lack of oxygen or necessary redox conditions will limit in-situ biorestoration
of contaminated soil and groundwater. When applying in-situ biorestoration in
practice, oxygen is usually the- limiting factor.
The alternatives to oxygen supply used in the projects visited were:
- air
- pure oxygen
- hydrogen peroxide
- nitrate
- nitrate / ozone
- methane / oxygen
Oxygen sources
The simplest method of supplying oxygen is aeration. However, the amount of
oxygen that can be added with air is strongly limited: only 8 mg/1 under
normal groundwater conditions (table 1). As a result, very large volumes of
oxygenated water may have to be infiltrated at the contaminated site, and
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because of permeability constraints, the remediation time is then relatively
long.
Table 1. Available quantities of oxygen from different sources.
air-saturated
water
02-saturated
water
H202
NO,
200 mg/1 200 mg/1
available oxygen
(me/1 at 10°C')
10
40
94
168
As shown in table 1, this problem can be overcome in part by using pure
oxygen (40 mg 02/1) or hydrogen peroxide (100 mg 02/1 from 200 ppm H202):
H202
H20
Hydrogen peroxide is toxic at higher concentrations and can therefore only
be used up to a limited concentration. In the case of H202, the
bioremediation is usually started with low concentrations (40-50 mg
H202/l), or even with pure oxygen. The objective of this measure is to let
the indigenous population of microorganisms acclimate to the oxygenated
environment. Once the population is acclimated, the peroxide concentrations
can be increased in increments of approximately 50 to 250 ppm in intervals
increasing from approximately one week to one month (U3), to achieve an
increased infiltration of oxygen. Such a gradual increase of peroxide
concentrations can be continued up to a concentration of about 1000 ppm
H202.
In the initial phase of biorestoration, the oxygen supplied is utilized by
the microorganisms in the vicinity of the infiltration point. When
contaminants in this area have been degraded, the oxygen can be transported
over larger distances, and biodegradation will then occur in an area,
further away from the infiltration point. This process continues until
oxygen breakthrough at the withdrawal wells.
An important aspect with respect to peroxide is its stability. As
remediation of the site progresses, the H202 must be carried increasingly
longer distances. This means that H202 must be stable in order to deliver
the oxygen to the area where it is needed. The decomposition of peroxide is
catalyzed by metals, such as iron and manganese. H202 can also be degraded
by the bacterial cell, with the enzyme catalase serving as the catalyst. On
the other hand, phosphate can stabilize hydrogen peroxide (Britton, 1985).
This is actually performed at demonstration projects. The form of phosphate
is mostly monophosphate. To reduce phosphate adsorbtion to the soil, a
combination of simple and complex polyphosphate salts can be used (Brown
et.al., 1986). The use of phosphate solutions is twofold: as a nutrient, it
also has a positive influence on the biodegradation when the original
concentration of phosphate is too low.
Nitrate as electron acceptor
Nitrate can serve as an electron acceptor. Comprehensive fundamental
research regarding the use of nitrate has been performed in West Germany
(Riss et.al., 1987). Here, laboratory research showed that nitrate can only
be utilized when a first phase with elementary oxygen has passed, and when
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the nitrate is present under anaerobic conditions. There has not always
been given satisfaction to these preconditions when applying nitrate for
in-situ biorestoration (see e.g. projects Nl and N4).
As is shown in table 1, one part of nitrate is equivalent to 0.84 parts of
oxygen. Take, for example, the oxidation of methanol:
1.5 02 + CH3OH -* C02 + 2 H20
NOg + 1.08 CHgOH + H+ -» 0.065 C6H7N02 + 0.47 N2 -I- 0.76 C02 + 2.44 H20
(Brown, 1989).
Until now, application of nitrate has only occurred in a few German states
and only incidentally in other countries. Application might encounter
licensing problems. In project N4, nitrate is added to the oxygenated
infiltrating ' water. Nitrate will also be used at project U3 as part of a
research program. Utilization of nitrate could not be determined in
research project Nl (Verheul et.al., 1988).
In project D9, a combination of ozone and nitrate has been used: ozone
above ground, to treat the water and oxygenate the organic contaminants;
nitrate in-situ, in the subsoil, to serve as an electron acceptor for
subsequent biodegradation by the microorganisms.
Co-metabolism -'-
At one research project (U5), biodegradation of chlorinated compounds by
methane-oxidizing bacteria (methanotrophs) involves stimulating the
population with methane- and oxygen-containing water. Hethanotrophs obtain
energy from the oxidation of methane. They synthesize the enzyme methane
monooxygenase, which catalyzes the first step in the oxidation of methane,
which they use for energy and growth. Monooxygenase oxidizes a range of
hydrocarbons, and appears to bring about the epoxidation of chlorinated
alkenes (co-metabolism):
CHC1-CHC1 + H20
CHC10CHC1 + 2H + 2e
These epoxides are unstable in water and hydrolyze to a variety of products
which can be oxidized readily by other heterotrophic bacteria to inorganic
end products (McCarty et.al., 1989, Janssen et.al., 1987).
Comparison of oxygen sources
Table 2 shows a comparison for various oxygen systems for a severely
contaminated site.
It can be concluded, that there is a wide range in both cost effectiveness
and in treatment effectiveness. For example, venting can only be applied in
the vadose zone. In terms of cost effectiveness, the order is:
venting » peroxide > nitrate > air sparger > water injection
while in order of treatment effectiveness, the order is
peroxide - nitrate > water injection > venting > air sparging.
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Table 2. Cost/performance comparison for various oxygen systems; high
degree of contamination.
Costs {$) Performance
System Capital Operation Maintenance kg/Day $ Site Utilization Tine of S/kg oxygen
Oxygen Treated Efficiency % Treatment Used
Air Sparging
Water Injection
Venting System
Peroxide System
Nitrate System
35
77
88
60
120
,000
,000
,500
.000
,000
800/nonth
1200/roonth
1500/month
10.000/month
6500/month
1200/month
1000/raonth
1000/month
1500/month
1000/month
3
4
1810
86
96
41
75
60
100
100
70
50
5
15
12.5
858
1580
132
330
335
days
days
days
days
days
57.
62.
8.
41.
49.
-
-
-
-
-
(Brown, 1989)
The choice of an oxygen supply is most dependent on the contaminant load,
the mass transfer and the ease of transport and utilization. At low
concentrations, simple systems, such as air sparging, become more cost
effective.
Nutrient supply
The biodegradation rate will be limited when inorganic nutrients, such as
nitrogen and phosphorus, are present in limiting concentrations or mutual
ratio's. Regarding contaminated sites, the presence of nitrogen and
phosphorus should be viewed in relation with the carbon concentration from
the contaminants. In soil, a C:N:P ratio of 250:10:3 is considered to be
optimal for biodegradation. Also other C:N:P-ratios, e.g. of 100:10:2 have
been chosen.
The need for nutrients is dependent on the site characteristics. At certain
sites, nutrient addition can be unnecessary. In other cases, increasing the
inorganic concentrations at one time can be sufficient. If nutrient supply
is needed during the clean-up, nearly always batch-mode addition has been
chosen.
In order to satisfy nutrient requirements, a wide range of components can
be added. This includes compounds like NH4N03,-Na- and K-orthophosphate and
trace elements.
In a few projects, such as Nl, addition of an easy degradable carbon source
(NaAc) enhanced the initial degradation of hydrocarbons during laboratory
experiments. However, the significance for demonstration scale seems to be
limited.
Addition of mlcrobial populations
Besides stimulating the indigenous microbial population to degrade organic
compounds in the subsurface, another option is to add microorganisms with
specific metabolic capabilities to the subsurface. This is demonstrated in
projects D3, D4 and D7. Soil samples are taken from the contaminated site
at spots where microorganisms occur, for example at the edge of the
contamination. The microorganisms which are present at those spots, will be
adapted to the contamination in the soil, and will be able to degrade the
contaminants. The samples are taken to .the laboratory, where selection
occurs by enrichment culturing, until a suspension is obtained which
contains the selected microorganisms in high density. This suspension of
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microorganisms is then injected with the infiltrating water at the
contaminated site. The objective of this inoculation is to increase the
number of adapted microorganisms at the site, in order to accelerate
biodegradation.
Another method to add microorganisms to the subsoil is applied when a
biological groundwater treatment plant is applied above ground (N2 and N4) .
The effluent of such an installation will contain large amounts of adapted
hydrocarbon degrading microorganisms which are injected in the soil. In a
research project, also inoculation by effluent water of a wastewater
treatment plant was used (project N5).
However, there is much uncertainty about the efficacy of the addition of
microorganisms to the subsoil and the possibilities of transporting
bacteria through the soil, in order to get them at the spots where they are
needed. Generally, 95% of the soil population tends to adsorb on soil
particles, whereas only 5% can be transported.
Results of the in-situ biorestoration projects
The projects visited differ widely in the clean-up results to be obtained.
For example, some projects do not aim to achieve a given concentration; on
the basis of the clean-up progress, it is decided what the residual
concentrations of contaminants should be.
There are much differences; in the Netherlands, the objectives set by the
legislator are generally 50 /jg mineral oil per liter (groundwater) and 50
mg/kg in the soil. In a few cases, this level can be 200 pg/1 and 1000
mg/kg d.w. in the soil.
In relation with the residual concentrations, it is important to notice
that different objectives have been used; in several projects, the goal was
to reach low concentrations in the groundwater, whereas in other projects
low concentrations in the soil were decisive.
In the USA and West Germany, the reported objectives varied between
undetectable levels (mineral oil) in the groundwater and less than 5000
mg/kg in the soil.
As regards results of in-situ biorestoration, the visited projects can be
divided into three groups:
a) demonstration projects that have been finished (N3, N5, D4, D7, D8, D9,
Ul, U6)
b) demonstration projects that are under way (N2, N4, Dl, D2, U2, U3, U4,
U5, U7)
c) projects which are in the stage of preparation of a field demonstration
(Nl, D5, D6).
This paragraph is limited to projects of groups a) and b). Table 3 shows
the results of the finished projects.
About half of the in-situ biorestoration projects reviewed in this study
have been finished.
The significance of the results appears from relating the residual
concentrations, which have been reached in the projects finished, to the
-------
- 13 -
corresponding concentrations of the Netherlands examination framework for
soil pollutants.
Table 4 shows the relevant values of this framework.
Table
3. Residual concentrations and remediation time
biorestoration projects finished.
for a few in^situ
Code
N3
N5
D4
D7
D8
D9
Ul
U2
U6
Contaminant
aromatics
mineral oil
BTX
diesel oil
diesel oil
fuel oil
diesel/arom.
aromatics
oil
gasoline
4-chloro-2-
me thy Ipheno 1
JP 4
Residual
concentration
< 30 /ig/1
< 200 Mg/1
< 5 mgAg
150 mgAg
4600 mgAg
< 100 mgAg
30 mgAg
non- detectable
levels
< 10 mgAg
> 80% of area
cleaned-up
550 kg he removed
Compartment
water
water
soil
soil
soil
soil
soil
water
soil
water
~ ~
Remediation time
("months')
6
12
18
12
9
3Xv
10 '
48
24
12
x) non-detectable levels reached for part of the contaminated area; clean-
up was continued for gaining complete clean-up of the site.
Table 4. Relevant part of the Netherlands examination framework for soil
pollutants.
Indicative values: A - reference value
B - indicative value for further investigation
C - indicative value for cleaning-up
Presence in:
soiKmg/kg dry weight)
A B C_
groundwater (/tg/1)
A B C
benzene
ethyUtenzene
toluene
zylene
phenols
aromatics (total)
polycycllc araBatic
total FAHs
flhlmHim*— » »-B— «««
aliphatic chlor.
comp. (total)
chlorophenols
(total)
mineral oil
0.05(d)
O.OS(d)
O.OS(d)
0.05(d)
O.OS(d)
-
Uydrocajjbo
1
T
-
*
0.5
5
3
5
1
7
am IfOft
20
7
1
1000
5
SO
30
SO
10
70
200
70
10
5000
0.2(d)
0.2(d)
0.2(d)
0.2(d)
0.2(d)
-
-
_
-
50(d)
1
20
IS
20
15
30
10
IS
0.5
200
5
60
SO
60
50
100
40
70
2
600
* • reference value soil quality
d • detection limit
-------
- 14 -
As regards the Netherlands examination framework for soil pollutants,
residual concentrations below B-level, or even undetectable levels of
contaminants have been reached in the finished projects. Five out of nine
projects reach the A-level, thus meeting the standards used in the
Netherlands. Venting (N5) was successful as regards volatile components
(petrol), but not for PAHs.
The results of the projects which are underway are generally promising.
Comparison of the results of the different in-situ biorestoration projects
is very tricky. This is mainly caused by the application of different
methods, used in the course of the in-situ biorestoration projects:
- methods of soil and groundwater sampling and analysis,
- determination of physical and chemical site parameters.
Occasionally, tht.re are also gaps in the total overview of the restoration
course.
The total overview of the in-situ biorestoration projects, as presented in
the appendix, also shows the results in relation with other aspects, such
as soil structure, oxygen source, applied system and nutrients used.
The remediation time varied between 90 days and 4 years, and is largely
dependent on the site characterization (soil structure) and the kind of
contaminants.
Costs for in-situ biorestoration
A wide range of site- and system characteristics and objectives influences
total costs for in-situ biorestoration projects. These include:
- geology and soil structure
- type and concentrations of contamination
- distribution of contaminants in the subsoil
- total surface and volume of the contaminated area
- system characteristics: recirculation, water and gas treatment a.o.
Because these aspects can vary significantly, the costs for completing the
projects can vary considerably.
It must be stressed that these figures should always be seen in relation to
other treatment techniques for a certain contaminated location, including
cost for excavation and transport.
The projects can be divided into two main groups:
- petrol stations (approximately 400 - 1,000 m ; 1,000
- refinery- aijd industrial sites (approximately 20,000
- 400,000 m ).
Petrol stations
5,000 m i
75,000 m
30,000
Costs for in-situ biorestoration at contaminated petrol stations varied
between 62,000 and 750,000 US $ (40- 250 ys $/m ). Included are relatively
cheap projects of approximately 60 US $/m which could be performed without
abovegroundwater treatment (Ul) or without water recirculation (D4).
-------
- 15 -
A comprehensive itemization of the different costs for using in-situ
biorestoration to treat a specific petrol station is shown in table 5
(Fournier, 1988).
It should be noted that, dependent on the situation, the contribution of
hydrogen peroxide to the total cost of the operation can be substantially
higher; contributions of 90% have occurred.
Table 5. Estimated costs for in-situ biorestoration of a petrol station
(Fournier, 1988).
Capital costs
Groundwater monitoring wells 5,000
Reinjection well
Nutrient and peroxide addition equipment
Recirculation equipment
EquipMnt total
PraliMinaxy site) assasraent costs
Laboratory tests
Field tests
Reports
Total pre)l$MixuuT teM^*-ii>g
Total initial expenditure*
Groundwater monitoring
Reinjection well maintenance
Chemical costs
Total ••""•»T costs
Present worth factor for 3 years
Present worth of OfiM costs
Present worth of ISB option
Total costs error 3 Tears
S
2,300
5,000
5.000
17,300
16,300
5,000
2.000
23,300
40,600
8,200
14,200
12.000
34,400
X 2.402
82,630
123,230
£143.800
Z
12
16
72
_122
Refinery- and industrial sites
Cost for in-situ biorestoration at refinery- and industrial sites varied
between 330,000 and 16 millioj US $. Again, especially system design
determines total cost: 7.- US $/m if a relatively simple in-sijfu type of
landfarming is used (D2) up to approximately 150.- US $/m for a more
complex system design.
From the information from the projects it can be concluded that operating
and maintenance costs account for about 2/3 of the total costs. Generally,
1/3 of the costs is due to preliminary research and installation costs, in
about equal amounts.
In many cases in-situ biorestoration will be more cost-effective than other
techniques, such as incineration and soil washing of the excavated soil,
possibly combined with groundwater treatment (approximately 70-170 $/m
excluding excavation and transport costs (Staps, 1989a)).
-------
- 16 -
CONCLUSIONS
Application
- The locations at which in-situ biorestoration has been used can be
divided into two main groups:
* filling stations (service stations, airforce bases, marshalling yards,
bus stations) with leaking pipelines or storage tanks (400 - 1,000 m2),
* chemical industry sites, mainly (former) refineries (20,000-75,000 m ).
- With respect to soil structure and geology, nearly all locations can be
defined as sandy. Clay layers are present in several areas. Only in an
exceptional case in-situ biorestoration is used at a site with overburden
clay and fractured bedrock.
- Regarding hydrology, permeability is a very important parameter for in-
situ biore§toration8 In the projects reviewed, the K_-yalue5varied
between 10 and 10 m/s, but was mostly of the order of 10 -10 m/s.
In general, a K--value of 10 m/s is regarded as being the minimum
permeability required for successful application of in-situ
biorestoration.
- All locations were contaminated with hydrocarbons. Most contaminations
are defined as petrol and/or diesel. A few locations were contaminated
with PAHs or a mixture of chlorinated hydrocarbons, mineral oil and PAHs.
The frequent discovery of secondary sources of contamination points out
that the characterization is not always sufficiently carried out.
Design
- The approach of in-situ biorestoration at the visited projects could be
characterized by either a hydrological or a microbiological background.
Only rarely, a good integration of both disciplines could be seen.
- The decision for application of in-situ biorestoration can only be taken
after a comprehensive s ite-characterization. The specific
characterization of the contaminated site and preliminary biotreatment
laboratory studies (if possible followed by field studies) should be
performed to determine optimal stimulation actions and thus the
different forms in which the technology can be applied.
- As regards hydrological measures. generally a system is designed, in
which the groundwater is centrally withdrawn and, after aboveground
treatment, is reinfiltrated at several spots at the outer border of the
location. In order to support the degradation in the subsurface, an
aboveground treatment system is used to degrade the contaminants in the
groundwater which is pumped-up, and to condition the water before
reinfiltration.
- As regards the aboveground treatment, the first part is generally a
sandbox. Undissolved contaminants are removed in an oil/water separator.
An air stripper is applied for removal of volatile contaminants. At a few
projects, biological systems, such as a trickling filter, were used for
degradation of dissolved compounds.
-------
- 17 -
- Recirculation of the pumped-up groundwater has positive effects on the
biodegradation in the soil. This may be due to the infiltration of
degradation products, which are relatively easy to break down and which
stimulate the activity of the microorganisms in the subsoil.
- The contaminating vapours in the air from the air stripper can be
oxidated by means of a biological compost filter or a catalytic oxidizing
system in order to acquire degradation of the contamination instead of
moving the contaminants from one compartment (groundwater) to another
(air).
- On demonstration scale, most of the time the limiting factor is lack of
oxygen or necessary redox conditions. Hydrogen peroxide is most popular
as oxygen source. However, for certain applications it can be relatively
expensive. Other sources are air, pure oxygen and nitrate (as electron
acceptor). The choice for a system is based on cost-efficiency,
contaminant load and the ease of transport and utilization.
- Necessary nutrient addition is fully dependent on the original available
nutrients in the soil and the uptake by the microorganisms. Usually,
addition of nitrogen and phosphorus is necessary. In a few cases, also
trace elements have been supplied. Other projects could be biorestorated
without any artificial supply of nutrients.
- The effect of the adding detergents is still questionable. Fundamental
research and most practical experience indicate that the effect on
degradation is negative. Clogging of the soil can occur when detergents
are supplied, probably due to an interaction between the oil, water,
detergent and solid phase.
- Addition of microorganisms to the subsoil, with the aim of enhancing the
biodegradation, is being used by a few companies. Although such supply
will always have some beneficial effect, until now, this has not been
proved. Cost-benefit calculations are also lacking. A major objection
here is, that soil microorganisms tend to adsorb onto (soil) particles,
and consequently cannot be transported over long distances in the
subsoil. This implies that the effect of the inoculation is very limited.
White spots
- Bottle-necks in relation with in-situ biorestoration can be:
* insufficient infiltration rates, mostly caused by clogging,
* insufficient hydrological isolation,
* relatively long remediation period, needed for reaching low
concentrations of contaminants,
- When using in-situ biorestoration, the precise fate of degraded
hydrocarbons. such as gasoline, is not yet known. A proportion is
transformed to leachable DOC, another part to DIG, but a large part is
still unaccounted for.
- With the exception of project Nl, research on in-situ biorestoration has
not provided knowledge about mass balances. When degradation occurred in
project Nl, the percentages of leached and degraded aromatics were about
-------
- 18 -
the same. The aliphatics were removed by degradation only, and then
almost completely.
Results and significance
- As regards feasibility, in-situ biorestoration can technologically
compete with other technologies when it is applied at a suitable
location, and the process is well run. As regards the Netherlands
examination framework for soil pollutants, residual concentrations below
B-level, or even undetectable levels of contaminants have been reached in
most of the finished projects. Contaminants are mainly hydrocarbons
(gasoline, diesel, mineral oil).
The remediation time varies roughly between 3 months and 4 years, largely
depending on the initial concentrations, the kind of contaminants, the
soil structure and the requirements which are set. Concerning practical
projects 3without research aspects, costs can vary between approximately
40-80 $/m . This means that in many cases in-situ biorestoration will
alsg be more cost-effective than other techniques (approximately 70-170
$/m excluding excavation and transport costs (Staps, 1989 )).
RECOMMENDATIONS
General policy
- This evaluation included the visit of 17 contaminated sites, and
concludes that in-situ biorestoration is a promising technology for a
selection of contaminated sites. However, it is important to notice that
most spills, and thus damage to the environment and the spending of large
amounts of money for remediation, could have been prevented by good
house-keeping. Therefore, at locations where spills might occur,
prevention is recommended in the first place.
- The most fundamental recommendation that can be made from this study, is
to stimulate the development of in-situ biorestoration. This study shows
that the technology has a large potential. At present, it is important to
collect reliable (demonstration) data, which can be used in the following
areas:
* optimization of the technology, mainly regarding oxygen transport and
utilization, peroxide transport and stability and removal of
contaminant residuals from soils (bio-availability).
* extending the technology's range of applications, especially to more
recalcitrant contaminants.
* development of models of (in-situ) biorestoration.
- In-situ biorestoration is expected, to be a promising technology,
especially for application at contaminated industrial sites. This is
mainly because of the minimal physical impact on the environment, caused
by the process; industrial activities can be continued during the clean-
up.
- When demanding certain residual concentration levels, regulators should
not only consider concentrations in the groundwater, but also in the
soil. It should be prevented that an in-situ biorestoration project is
finished because the contamination levels in the groundwater are
-------
- 19 -
sufficiently low, while significant concentrations are still present in
the unsaturated zone of the soil. Percolating water from precipitation
will transport (a part of) residual contaminants and contaminate the
clean groundwater again, making a second clean-up operation necessary.
- The approach taken by the experts involved in several of the projects
visited can be characterized by either a hydrological or a
microbiological background. However, in-situ biorestoration is not only
pure biotechnology, but is indeed an integration of biotechnology and
hydrology. Integration of a number of disciplines is indispensable.
- Because of the general complexity of soils, the course of the degradation
process can never be predicted completely. Therefore, preliminary
research, both in the laboratory and in field tests will always be
necessary. The field tests should include oxygen utilization rates,
possible in-situ peroxide stability and potential clogging problems.
Laboratory methods for predicting the course of the in-situ
biodegradation should all be improved.
- There is a need for more sharing of meaningful site data by those
experiencing in this technology. This is especially needed as regards
data on peroxide stability and transport, oxygen utilization and the
removal of fuel residuals from soils. Therefore, projects like Nl, U3 and
U6 are very useful. An open policy of organizations with experience of
the technology can expose bottlenecks concerning both practice and
demonstration, thereby directing the research of universities and
institutes and making this research more valuable.
- Knowledge about modelling of transport behavior in the soil seems to be
sufficient. Modelling of biodegradation processes in the soil however, is
still a difficult problem and requires further attention. A precondition
for further development however is the availability of representative
data, which should be published by the experts involved in in-situ
biorestoration projects.
System design
- Venting of volatile contaminating compounds in the unsaturated zone and
treatment of these components above ground (possibly combined with
recirculation and biorestoration in the saturated zone) seems to be a
promising and cost-effective method calling for further attention.
- A combination of chemical treatment above ground and biological treatment
in the subsoil can possibly expand the application of in-situ
biorestoration, especially to compounds which are more difficult to break
down biologically (such as PAHs) and more readily biodegraded once a
first oxidation step has taken place. Further research in this field can
be recommended.
- Stimulation of the biological activity by heating the infiltrating
groundwater was used at one project only (D5). Here, it was not
conclusively shown that this was a cost-effective method. Measurements in
test plots should be conducted to demonstrate whether and when the
heating effect is economical.
-------
- 20 -
- There is much uncertainty about the efficacy of the supply and
distribution of oxygen (-sources) in the subsoil. Research_on alternative
oxygen sources (02, H202) and electron acceptors (N03) is useful.
Hydrogen peroxide is a relatively expensive oxygen source, the more so
because only a very limited part of it can actively be used for the
biodegradation of the contaminants; this is estimated to be approximately
15% (Brown, 1989).
- In-situ peroxide stability must be greatly improved to provide adequate
oxygen downgradient of injection points.
- As regards inoculation, the selection by enrichment culturing is
especially performed by compounds of the contamination. A very
interesting possibility would be to expand this technique to a selection
for the tendency of microorganisms to adsorb onto soil particles. The
small percentage of the population that does not tend to sorb, could thus
be selected, possibly resulting in improved biodegradation in situ
because these organisms can be carried a longer distance in the subsoil.
This aspect needs further attention.
- Co-metabolism, such as the biodegradation by methanotrophes, deserves
more attention because it may broaden the applicability of
biorestoration.
- Detergents could be useful with respect to the following aspects:
* limitations caused by the low availability of contaminants to the
microorganisms,
* extension of the applicability of in-situ biorestoration for compounds
with a low solubility.
In order to open up possibilities for these aspects, fundamental research
into the use of detergents in this field is necessary. Not only
artificial supply of detergents in the in-situ biorestoration system
should be considered, but also the possible use of surfactants produced
by microorganisms in the soil.
Mass balances
- There is a strong need for mass balances on both laboratory and pilot
plant scale. Mass balances will improve the insight in the contribution
of different processes in the total biodegradation process.
- The limited possibilities to monitor biological activities in the soil is
partly responsible for the lack of knowledge about the process of in-situ
biorestoration. The development of methods, which can be used for
monitoring the biological processes in the soil, would greatly contribute
to a better understanding of the processes, and thereby, to a more
selective and economical supply of for example oxygen and nutrients.
- In order to gain a better insight into the contribution of biodegradation
to the total degradation process in the laboratory, a satisfactory method
for sterile experiments should be developed. The methods currently
available are insufficient.
-------
- 21 -
- The precise fate of degradation products is not yet known. A proportion
is converted to leachable DOC, another part to DIG, but a large part is
still unaccounted for. Insight into the quantity, quality and
significance of degraded hydrocarbons, such as gasoline, is needed,
especially as regards the question of "how clean is clean?".
Specific problems
- More attention should be paid to the problem of clogging in the subsoil,
resulting in disappointing infiltration rates. This problem can be
related to different factors, such as geology (permeability), excessive
growth of microorganisms, or high concentrations of iron or manganese.
- Once relatively low residual (threshold) concentrations with in-situ
biorestoration have been reached, the limiting factor usually becomes the
availability of contaminants to the microorganisms. This is in the region
of, for example, less than 250 mg/kg of dry soil in the case of mineral
oil. When cleaning up soil contaminated by mineral oil in the
Netherlands, residual concentrations must always be less than 50 mg/kg.
This makes the limiting factor in this case, principally availability,
even more important. Further fundamental research in this area is
recommended.
Overview
An overview of the most important recommendations is given in table 6.
\
Table 6. General overview of recommendations.
|Policy
(System design
(Research
stimulation of experience
end (hexing of information
integration of microbiology,
hydrology and (soil-)
chemistry
• preliminary research
including heating and
mass balances
' consideration of both
soil and groundwater
* combination of bioresto-
ration and venting-
* problem of clogging
* oxygen:
- supply and distribution
- alternative oxygen
sources
- peroxide stability
* monitoring possibilities
* extension to broader
application
* threshold concentrations
* eo-aetabolism
* addition of micro-organisms
•addition of detergents
* sterile experiments
* modelling of biorestoration
* combination of chemical ~-
and biological treatment
-------
- 22 -
ACKNOWLEDGEMENTS
The author wishes to acknowledge the experts visited who are involved in
in-situ biorestoration projects. Without their contribution, this
evaluation could never have been made. The open discussions with many of
these experts gave considerable support to this report.
The author wishes to thank Mr. Donald Sanning of US-EPA Cincinnati,
director of the above mentioned NATO/CCMS Pilot study, who has played an
important role in contacting key experts in in-situ biorestoration in the
USA.
LITERATURE
Brown, R.A. Oxygen sources for biotechnological application. Paper
presented' at Biotechnology Work Group. Feb. 21-23, 1989, Monterey,
California.
Brown, R.A. , Norris, R.D. and Westray, M.S. In situ treatment of
groundwater. Presented at HAZPRO '86, The Professional Certification
Symp. and Exp., Baltimore, Md., April 1986.
McCarty, P.L., Semprini, L. and Roberts, P.V. Methodologies for evaluating
the feasibility of in-situ biodegradation of halogenated aliphatic
groundwater contaminants by methanotrophs. Proceedings, AWMA/EPA
Symposium on biosystems for pollution control, Cincinnati, Ohio, Feb.
21-23, 1989.
Downey, D.C. Enhanced Biodegradation of jet fuels. Eglin AFB, USA. A Case
Study for the NATO/CCMS Pilot Study on Remedial Action Technologies for
Contaminated Land and Groundwater - November 1988.
Eyk, J. van and Vreeken, C. Venting-mediated removal of hydrocarbons from
subsurface soilstrata as a result of stimulated evaporation and enhanced
biodegradation. Proceedings of Forum for Applied Biotechnology. The
Faculty of Agricultural Sciences. State University of Gent, Belgium.
Gent, September 29, 1988.
Fournier, L.B. An effective treatment for contaminated sites. Hydrocarbon
Technology International, 1988, p. 207-210. Sterling Publishers, London.
Janssen, D.B., Grobben, G. and Witholt, B.H. Toxicity of chlorinated
aliphatic hydrocarbons and degradation by methanotrophic consortia. In:
Neijssel, O.M., Meer, R.R. van der and Luyben, K.C.A.M. (Eds.)
Proceedings of the fourth European Congress on Biotechnology, Vol. 3.
Elsevier Science Publishers, Amsterdam. 1987.
Lee, M.D., Thomas, J.M., Borden, R.C., Bedient, P.B., Wilson, J.T. and
Ward, C.H. Biorestoration of aquifers contaminated with organic
compounds. CRC Critical Reviews in Environmental Control, Volume 18,
Issue 1 (1988), p. 29-89.
Nagel, G. , Kuehn, W. , Werner, P. and Sontheimer, H. Sanitation of
groundwater by infiltration of ozone treated water. GWF-
wassser/abwasser, 123 (8): 399-407, 1982.
Riss, Gerber and Schweisfurth. Mikrobiologische Untersuchungen ttber
wesentliche Faktoren bei der unterirdischen Beseitigung organischer
Altlasten unter anaeroben Bedingungen mit Nitratdosierung. Universitat
des Saarlandes, Homburg/Saar. 1987.
Socz6, E.R. and Staps, J.J.M. Review of biological soil treatment
techniques in the Netherlands. In: Wolf, K., van den Brink, W.J. and
-------
- 23 -
Colon, F.J. (Eds.), Contaminated Soil '88, p. 663-670. Kluwer Academic
Publishers, 1988.
Staps, J.J.M. European experience in hydrocarbon contaminated groundwater
and soil remediation. RIVM-report no. 738708002. 1989a.
Staps, J.J.M. International evaluation of in-situ biorestoratign of
contaminated soil and groundwater. RIVM-report no. 73708006. 1989 (in
press).
Verheul, J.H.A.M., van den Berg, R. and Eikelboom, D.H. In situ
biorestoration of a subsoil, contaminated with gasoline. In: Wolf, K.,
van den Brink, W.J. and Colon, F.J. (Eds.), Contaminated Soil '88, p.
705-716. Kluwer Academic Publishers, 1988.
-------
-------
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