311
Bioaugmentation as a soil bioremediation approach
Timothy M Vogel
The debate over the efficacy of bioaugmentation rages on,
with research continuing to demonstrate that its advantages
for soil bioremediation are difficult to predict; however,
when it works, the results are often very encouraging. The
difficulties arise from, among others, the diversity of the
microorganisms used, environmental heterogeneity, and
variations in the influence of critical parameters (e.g. humidity,
microbial predation and 'bioavailability') which, unfortunately,
are not even always identified.
Address
Rh6ne-Poulenc Industrialisation, 24 avenue Jean-Jaur6s, 69153
D~cines-Charpieu, France; e-mail: TIMOTHY.VOGEL@univ-lyonl .fr
Current Opinion in Biotechnology 1996, 7:311-316
© Current Biology Ltd ISSN 0958-1669
Abbreviations
PAH polycyclicaromatic hydrocarbon
PCP pentachlorophenol
Introduction
Bioaugmentation, the addition of microorganisms to en-
hance a specific biological activity, has been practiced
intentionally for years in a number of areas, including
agriculture and forestry [1] and wastewater treatment [2].
Research attempting to evaluate the value of bioaugmen-
tation for soil remediation is not new either (e.g. [3,4]).
Bioaugmentation is not generally accepted as an efficient
technique for soil bioremediation, although proponents
continue to demonstrate possible advantages. It is often
viewed negatively by those either who have had cautionary
experiences or worse (e.g. the addition of bioaugmentation
products either decreased biodegradation rates or clogged
aquifers) or who do not believe that the advantages of
increasing the biocatalyst activity offset the advantages of
niche fitness demonstrated by indigenous microorganisms
[5-8]. T h e utility of bioaugmentation is supported by
studies showing the incompetence of indigenous micro-
organisms in some cases and the apparent enhanced
bioremediation rate after the addition of competent
microorganisms [9,10",11,12,13°]. The reinoculation of soil
with indigenous microorganisms directly isolated from the
same soil is often included in the term bioaugmentation
[14,15].
With the wealth of experiments that demonstrate the
potency and problems of bioaugmentation, several critical
factors have become evident [2,16,17]. Increased interest
in this area is highlighted by the recent publication of a
book entitled Bioaugmentation for Site Remediation based on
conference presentations [18]. Certainly, today, researchers
can design experiments to either succeed (or not); what
is at issue is the way in which these experiments are
performed and the associated parameters yet to be dis-
covered or understood. Among the parameters that appear
to be important are the pollutant characteristics (e.g.
bioavailability [19,20], concentration [21] and microbial
toxicity [22]), the soil physico-chemical characteristics (e.g.
humidity or water content [23], organic matter content
[23], clay content [23] and pH), microbial ecology (e.g.
presence of predators [24] and interspecies competition),
microbiology (e.g. the presence of co-substrates [25],
genetics of the relevant organisms, and enzyme stability
and activity [26]), and methodology (e.g. inoculation
concentration [27,28] method of inoculation [28,29], the
presence/absence of indigenous activity [9,30], and in-
oculum heterogeneity [31]). Therefore, when individual
attempts at bioaugmentation research are reported, the
difficulty in characterizing, quantifying, and evaluating all
of the potential parameters leads to general conclusions
that might not be widely acceptable.
This review discusses the above parameters; the lack of
current understanding on the relative importance of these
parameters demands an intuitive approach to a certain
extent. The rationale for the use of bioaugmentation
is the perceived inability of indigenous microorgan-
isms to perform satisfactorily during the bioremediation
of contaminated soil. This 'failure' is categorized and
characterized differently depending on the chemicals
to be degraded, time of treatment, etc. Thus, the
opinion that bioaugmentation is better or worse than
bioremediation using indigenous microorganisms is always
relative! Nonetheless, a review of the literature concerning
the factors responsible for the performance of indige-
nous microorganisms to bioremediate a contaminated
soil (biostimulation) would find many issues in common
with this one (e.g. potential additives for bioremediation
enhancement [32]).
Recent literature (1994-1995) contains a high percentage
of articles concerning the injection, tracking and fate of
added microorganisms during bioaugmentation, with a
smaller number of articles concerned with their effective-
ness once in the ground. This apparent emphasis is the
result, at least in part, of the application of molecular
biology techniques such as gene probes.
Indigenous microorganisms
T h e adaptation capacity of indigenous microorganisms is
currently under study by several groups, and suggestions
are starting to be made that this capacity is tremendous
and perhaps more rapid than previously thought. For
example, environmental stress might enhance mutation
rates (soil environments are comparatively more harsh than
laboratory conditions). As these rapidly adapting microor-
ganisms are going to be the reference for bioaugmentation,
312 Environmentalbiotechnology
consideration should be given to their applicability to
the bioremediation of contaminated soil. T h e y are often
better distributed, in general, than added microorganisms,
although not necessarily with regard to the target pollutant
because both the pollutant and the added microorganisms
often enter the soil by similar methods ('dumping'). One
important concept is, thus, related to the distance between
the target compound and microorganism. Perhaps added
microorganisms are closer to compounds recently 'added'
and indigenous microorganisms are closer to older 'historic'
pollution, although concepts such as size exclusion [31]
might be important determinants of microbe-compound
proximity.
Are indigenous microorganisms more likely to be found
within soil aggregates than 'added' microorganisms? Are
recently 'added' chemicals to be found outside aggregates
near added microorganisms? Are soil surface characteristics
going to influence temporal distributions of compounds
and microorganisms? Answers to these questions depend
in part on recent techniques such as the use of gene
probes. Genetic techniques can be used not only to
monitor the presence of specific microorganisms [33,34],
but also to monitor the contact between compound and
microorganism using luminescence genes [35,36",37].
Compound characteristics
Pollutant toxicity is often used as justification for bioaug-
mentation because, conceptually, this toxicity could inhibit
the degradative activity of indigenous microorganisms.
Although few sites with obvious toxicity have been
reported, the sites that have been described are of clear
potential for bioaugmentation if the added microorganisms
can also resist the toxicity. One site where exogenous
microorganisms were employed required dilution by soil
washing or bioslurry techniques to achieve pollutant
degradation [22].
Compound 'biodegradability', which is associated with
many factors, including those discussed below, is some-
times related to compound structure and its related
physico-chemical characteristics such as solubility and
bioavailability (which itself is not intrinsic to the com-
pound, but related to the interactions between the
compound, the microorganism(s) and the soil). Curiously,
reports about the limited use of bioaugmentation relative
to biostimulation often study compounds that are either
known for their 'non-availability' (e.g. low concentrations
of polycyclic aromatic hydrocarbons [PAHs] [20]) or rela-
tively easy to degrade when other limiting conditions (e.g.
nutrients) are provided such as petroleum hydrocarbons
[6,7] or crude oil [38]. Even so, for compounds that are
considered relatively recalcitrant but generally 'available',
bioaugmentation has been demonstrated to be beneficial
(e.g. 2,4,6-trinitrotoluene [9], carbon tetrachloride [39,40],
and pentachlorophenol [PCP] [41,42]). Care must be
taken not to conclude too much from such data as
'adapted' microorganisms are frequently found in soils
contaminated with compounds of low bioavailability.
Clearly, our understanding is limited of the mechanisms
of adaptation regarding critical initial concentrations of
pollutants.
Physico-chemical environmental
characteristics
Environmental conditions play a pivotal role in deter-
mining biological activity, whether of indigenous micro-
organisms, added microorganisms, or cultured indigenous
microorganisms returned to the soil. These conditions fall
into two general categories: those that reduce the microbial
activity, such as temperature, humidity and ionic strength
(which, in one report, also increased cell attachment
[43"]); and those that restrict the mass transfer (mainly by
diffusion) of the compound to the microorganism, such as
clay and organic-matter content [23]. In addition, several
aspects of the bioaugmentation process are affected by
advective transport, such as permeability. This affects the
addition of microorganisms during in situ bioaugmentation
as well as the addition of nutrient and electron acceptors
during both bioaugmentation and biostimulation.
As stated above, emphasis recently has been on the
understanding of the movement and fate of added mi-
croorganisms during bioaugmentation. Under uncontrolled
conditions, microorganisms added in situ via injection wells
can clog well-heads and lead to overall failure of the
bioremediation strategy [5]. Curiously, the relatively few
microorganisms that traverse considerable distances in the
subsurface are considered insufficient by those interested
in bioaugmentation--column studies have demonstrated
an average microbial concentration 10-fold or higher in
the first 5cm than elsewhere in the soil column [44].
Accordingly, most research focuses on both controlling
environmental conditions [43"] and controlling microbial
development (e.g. resuscitation of starved non-sticky
ultramicrobacteria [45]) to prevent microorganisms from
clogging well-heads, but also to induce them to attach in
sufficient numbers in the aquifer to improve contaminant
degrad)tion. Even small concentrations of microorganisms
are of concern to either those monitoring the spread of
fecal contamination [46] or those who believe that micro-
organisms can enhance the movement of contaminants in
a manner similar to colloids [47].
Niche adjustment
Although microbial ecology issues are among the most
important in bioaugmentation approaches, unfortunately,
they are rarely addressed. Parameters that could influence
the performance of added (or indigenous) microorgan-
isms include niche fitness (competition, synergy, etc.),
steady-state microbial concentrations, and predators. One
can demonstrate niche fitness in an inverse sense by
instituting in soils changes that favor the development
and performance of a particular added microorganism.
This is exemplified by the enhanced growth and activity
of both Pseudomonas sp. strain KC under slightly alka-

line conditions (pH8.2 [39]) and the white-rot fungus
Phanerochaete chrysosporium under slightly acidic conditions
(pH5 [6], pH4.5 [48] and p H 6 [491). Fitness for the
soil environment can be defined by the quantity of
microorganisms (i.e. development), but for t h e treatment
goals of bioaugmentation, performance is usually the
more important factor. An interesting example is where
one nutrient in excess and another in limitation both
lead to an improved performance [501. T h e addition
of nutrients to optimize the performance of an added
microorganism can also lead to the increased development
of indigenous microorganisms, which themselves either
aid the treatment process (biostimulation [7,38]) or hinder
the process by consuming the added nutrient or carbon
source (e.g. starch [8]). In cases where co-metabolism
is desired, the consumption of added substrate by
indigenous microorganisms incapable of co-metabolizing
the pollutant leaves little for the added microorganisms
and, thus, results in poor performance [25].
T h e difficulties in adjusting the environment or in
selecting microorganisms fit for their target environment
have led to the development of techniques for protecting
the added microorganisms such as encapsulation [51,52].
In general, these techniques improve the long-term
viability of added cells [51]. Real engineering performance
evaluation is hindered by the unrealistic experimental
design (e.g. the use of freshly added pollutant, mi-
croorganisms and laboratory conditions), but the initial
results are promising. T h e fate of added microorganisms
is not unrelated to the ecologically stable microorganism
concentration one observes in soils. Because the decrease
(or increase) in microbial populations tends toward an
asymptote, recent attempts to model this behavior follow
a certain logic [53°°]. T h e appealing aspect of this
model approach is the connection between growth/decay
and a natural ecological population density/concentration.
For example, the Eschetichia coli modeled had a final
natural concentration 8-30 orders of magnitude less than
the Pseudomonas species modeled [53°°]. T h e application
of this approach to encapsulated microorganisms and
to a range of pollutant concentrations would possibly
provide important insights into added microorganism
survival during bioaugmentation. In addition, an attempt
to connect natural levels of genetic 'aptitude', either from
indigenous or added microorganisms, to these models
would be stimulating.
Microbial ecology is very important in evaluating both
the potential success of bioaugmentation and its possible
advantages over biostimulation. Microorganisms are af-
fected by maintenance energy, the production of, and
resistance to, antibiotics and toxic metabolites, predation,
etc. T h e necessity for using 'long-lasting' microorganisms
in bioaugmentation, on the basis of the potentially slow
reaction kinetics (which are controlled by slow compound
desorption), is debatable. Improved understanding of the
microbial ecology, microbiology and genetics of competent
Bioaugmentation as a soil bioremediation approach Vogel 313
pollutant-degrading microorganisms has increased our
ability both to monitor the fate of added microorganisms
and possibly to discover 'better-suited' species or strains
for bioaugmentation.
Microbial ecology
The problems of monitoring microbial survival raised
above are now somewhat routinely addressed through
the use of gene probes [25], PCR technology [33,34]
or immunoassay technology [54",55"]. These techniques
provide a general absolute minimum detection limit of
100 microorganisms per gram of soil; thus, survival of an
exogenous microorganism after inoculation at concentra-
tions up to ten orders of magnitude greater (e.g. [27]), can
easily be monitored. Even so, detection by gene probes
and immunoassays does not provide evidence of microbial
activity (with the possible exception of mRNA probes)
and, therefore, our prediction of the performance of these
added microorganisms still lacks easy assessment. Indeed,
the fate of added microorganisms might not be related
to their activity, but that of the indigenous microflora
that have recovered relevant genetic material from the
inoculated species. This potential natural exchange of
genetic material is much easier to measure using recent
applications of molecular biology [56,57].
The above genetic approaches have also stimulated
research to improve the capacity of microorganisms
to degrade xenobiotic compounds [58,59]. T h e related
debate is whether these microorganisms are 'better' at
degrading difficult compounds than naturally 'trained'
microorganisms. 'Better' is defined not by microbial
numbers, but by performance, given the possibility to
biostimulate the indigenous population, as mentioned at
the beginning of this review. Performance has an economic
aspect, also. Some confusion is caused by the definition
of the problem in terms of the long-term effects of
bioremediation. Issues related to changes in the ecology,
the geochemistry, and the hydrogeology resulting from
either biostimulation or bioaugmentation are difficult to
specify. For example, suggestions regarding the use of
suicide genes to prevent long-term survival of added
microorganisms returns us to the definition of the 'best'
characteristics of added microorganisms. Microorganisms
that are fast-acting (although slow with chemicals that
are not rapidly available), short-lived (i.e. no long-term
'danger'), mobile (i.e. capable of penetrating into the
system), adhesive (except near the injection point to
avoid clogging), resilient (i.e. resistant to fluctuations in
pH, ionic strength, heavy metal concentrations, etc.) and
inexpensive with a wide range of degradative activity
represent the ideal for bioaugmentation.
Engineering process design
Given the uncertainties regarding the environmental
characteristics, microbial ecology and microbiology of
bioaugmentation, the lack of comprehensive engineer-
ing guidelines is understandable. Yet, the 'brute force'
314 Environmental biotechnology
approach has been shown to be relatively effective
in some situations. For example, experiments where
very high numbers of microorganisms--for example,
from 107 bacteria (g soil)-I [10 °] to 10 lz bacteria (g soil)-!
[ 2 7 ] - - h a v e been added to contaminated soils where
'bioavailability' is not particularly the limiting factor have
been relatively successful. T h e disagreeable aspect of
the 'brute force' approach is the lack of engineering
design. Engineering design requires an understanding of
the parameters that control the bioaugmentation process.
Good engineering needs to be applied to bioaugmentation
process design at the experimental level. Reports of
the advantages/disadvantages of bioaugmentation from
experiments where nutrients included in bioaugmentation
products have not been tested separately from the added
microorganisms [17], where the activity of indigenous
microorganisms have not been compared [59], etc., do
not help engineers determine the critical parameters
involved. Engineering experiments cannot, however, cover
all variables and, therefore, need to concentrate on the
critical parameters defined in part by microbiologists:
microbial inoculation quantity (with zero being an option),
technique (what form of inoculum, e.g. freeze-dried [29],
and with what nutrients) and then the parameters that
are the same for biostimulation (e.g. humidity, pH and
electron acceptor concentration).
Although geological characteristics, such as heterogeneity,
can pose daunting engineering hurdles to the use of
bioaugmentation for in situ bioremediation, the use of
added microorganisms in ground treatment is easier to
implement and justify. Bioreactors for the treatment of
groundwater are an example where bioaugmentation is
inapplicable, even if indigenous microorganisms selected
on the target contaminants are used. On the other
hand, soil treatment 'reactors' such as bioslurry systems
[15] or biopiles [13"] are easier to control and tend to
provide more convincing results when bioaugmentation
is compared with biostimulation. This does not discount
recent advances in in situ bioaugmentation, such as the use
of plants, their root systems and rhizospheric bacteria [60],
only that the heterogeneity is difficult to assess.
Finally, all of the above discussion has been made
independently of economic aspects other than those
relating improved rates to implicit cost reduction. Yet, the
decision of if and how to apply bioaugmentation really
should be more objectively based. As for biostimulation,
bioaugmentation has a calculable cost that will vary with
compound type, soil type, reactor type, and inoculum type
and quantity. As mentioned at the beginning of this article,
generalizations about the advantages of bioaugmentation
relative to other techniques can be easily disproven in a
given example. Careful decision-making processes need to
be employed to avoid misconceptions about fantastic or
frightful results.
Conclusions
Bioaugmentation clearly provides certain advantages over
biostimulation in cases where pollutant toxicity or a lack
of appropriate microorganisms (both quantity and quality)
are important. Determination of the potential success
of bioaugmentation requires an understanding of the
bioavailability of the pollutant, the survival and activity
of the added microorganism(s) or its genetic material,
and the general environmental conditions that control soil
bioremediation rates. T h e recent application of genetic
techniques has aided the comprehension of the fate of
added microorganisms, bringing us closer to identifying
the critical parameters for the engineering design of
bioaugmentation processes. Recent examples of both
successes and failures associated with bioaugmentation
provide a cautionary note to those who believe that the
debate is over.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•* of outstanding interest
1. Jasper DA: BioremediaUon of agricultural and forestry soils
with symbiotic micro-organisms. Aust J Soil Res 1994,
32:1301-1319.
2. RittmannBE, Whiteman R: Bioaugmentation: a coming of age.
Wat C)ual Int 1994, 1:12-16.
3. Portier R, Bianchini M, Fujisaki K, Henry C, McMilin D:
Comparison of effective toxicant biotransformation by
autochthonous microorganisms and commercially available
cultures in the in situ reclamation of abandoned industrial
sites. Schriftenr Ver Wasser Boden Lufthyg 1988, 80:273-292.
4. Pritchard PH: Use of inoculation in bioremediation. Curt Opin
Biotechno11992, 3:232-243.
5. Maxwell CR, Baqai HA: Remediation of petroleum hydrocarbons
by inoculation with laboratory-cultured microorganisms. In
Bioaugmentation for Site Remediation. Edited by Hinchee RE,
Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:129-137.
5. McGugan BR, Lees ZM, Senior E: Bioremediation of an oil-
contaminated soil by fungal intervention. In Bioaugmentation
for Site Remediation. Edited by Hinchee RE, Fredrickson J,
Alleman BC. Columbus: Battelle Press; 1995:149-156.
7. Neralla S, Wright AL, Weaver RW: Microbial inoculants
and fertilization for bioremediation of oil in wetlands. In
Bioaugmentation for Site Remediation. Edited by Hinchee RE,
Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:31-38.
8. M611erJ, Gaarn H, Steckel T, Wedebye EB, Westermann P:
Inhibitory effects on degradation of diesel oil in soil-
microcosms by a commercial bioaugmentation product.
Buff Environ Contain Toxicol 1995, 54:913-918.
9. Shin CY, Crawford DL: Biodegradation of trinitrotoluene (TNT)
by a strain of Clostridium bifermentans. In Bioaugmentation
for Site Remediation. Edited by Hinchee RE, Fredrickson J,
Alleman BC. Columbus: Battelle Press; 1995:57-69.
10. Briglia M, Middeldorp PJM, Salkinoja-Salonen MS: Mineralization
• performance of Rhodococcus chlorophenolicus strain PEP-1 in
contaminated soil simulating site conditions Soil Biol Biochem
1994, 26:377-385.
Demonstrates the influence of several operating parameters on the success
of PCP degradation by Ft. chloropheno/icus. Degradation rates increased
with increasing POP concentrations and microbial numbers; bioavailability

and soil humidity also played a role. The bacterium performed poorly in
saturated soils, although little difference in degradation rate was observed
between 50% and 80% of field capacity. A high level of organic matter (peat)
apparently reduced the PCP bioavailability.
11. Riggle D: Successful bioremediation with compost. Biocycle
1995, 36:57-59.
12. Dave H, Ramakrishna C, Bhatt BD, Desai JD: Biodegradation of
slop oil from a petrochemical industry and bioreclamation of
slop oil contaminated soil. World J Microbiol Biotechno/1995,
10:653-656.
13. LamarRT, Davis MW, Dietrich DM, Glaser JA: Treatment of a
• pentachlorophenol- and creosote-contaminated soil using
the lignin-degrading fungus Phanerochaete sordida: a field
demonstration. Soil Biol Biochem 1g94, 26:1603-1611.
A good example of small-scale field application of bioaugmentation, with
controls to establish the real benefits of the addition of microorganisms.
Application of the fungus Phanerochaete sordida to PCP-contaminated and
PAH-contaminated soil resulted in a relative increase in compound biodegra-
dation relative to controls. Three-ring PAHs were degraded further in non-
treated soil, whereas four-ring PAHs were degraded further in fungus-treated
soil. Five-ring and six-ring PAHs were not significantly reduced.
14. Phelps TJ, Siegrist RL, Korte NE: Bioremediation of petroleum
hydrocarbons in soil column lysimeters from Kwajalein Island.
Appl Biochem Biotechnol 1994, 45/46:835-845.
15. Otte M-P, Gagnon J, Comeau Y, Matte N, Greet CW, Samson R:
Activation of an indigenous microbial consortium for
bioaugmentaUon of pentachlorophenol/creosote contaminated
soils. Appl Microbiol Biotechnol 1994, 40:926-932.
16. Atlas RM: Bioaugmentation to enhance microbial
bioremediation. In Biotreatment of Industrial and Hazardous
Waste. Edited by Levin MA, Gealt MA. New York: McGraw-Hill;
1993:19-37.
1 7. ForsythJV, Tsao YM, Bleam RD: Bioremediation: when is
augmentation needed? In Bioaugmentation for Site Remediation.
Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus:
Battelle Press; 1995:1-14.
18. Hinchee RE, Frederickson J, Alleman BC (Eds): Bioaugmentation
for Site Remediation. Columbus: Battelle Press; 1995.
19. Harms H, Zehnder AJB: Bioavailabllity of sorbed 3-
chlorodibenzofuran. Appl Environ Microbio/1995, 61:27-33.
20. Rothmel RK, Gaudet JL, Schul WH, Shannon MJR,
Kishnamoorthy R, Smith JR, Unterman R: Biostimulation versus
bioaugmentation: two strategies for treating PCB-contaminated
soils and sediments. In Abstracts of the g4th General Meeting
of the American Society for Microbiology. Lss Vegas: ASM;
1gg4:#Q-153.
21. Aamand J, Bruntse G, Jepsen M, Jqrgensen C, Jensen BK:
Degradation of PAHs in soil by indigenous end inoculated
bacteria. In Bioaugmentation for Site Remediation. Edited by
Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:121-12?.
22. Baud-Grasset F, Vogel TM: Bioaugmentation: biotreatment
of contaminated soil by adding adapted bacteria. In
Bioaugmentation for Site Remediation. Edited by Hinchee RE,
Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:39-48.
23. Godbout JG, Comeau Y, Greet CW: Soil characteristics effects
on introduced bacterial survival and activity. In Bioaugmentation
for Site Remediation. Edited by Hinchee RE, Fredrickson J,
Alleman BC. Columbus: Battelle Press; 1995:115-120.
24. RamadanMA, EI-Tayeb OM, Alexander M: Inoculum size as a
factor limiting success of inoculation for biodegradaUon. Appl
Environ Microbiol 1990, 56:1392-1396.
25. Barriault D, Sylvestre M: Factors affecting PCB degradaton
by an implanted bacterial strain in soil microcosms. Can J
Microbio11993, 39:594-602.
26. Trombly J: Engineering enzymes for better bioremediation.
Environ Sci Technol 1995, 29:550A-564A.
27. Pearce K, Snyman HG, Oellermann RA, Gerber A:
Bioremediation of petroleum-contaminated soil. In
Bioaugmentation for Site Remediation. Edited by Hinchee RE,
Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:71-76.
28. Comeau Y, Greet CW, Samson R: Role of inoculum preparation
and density on the bioremediation of 2,4-D-contaminated
Bioaugmentation as a soil bioremediation approach Vogel 315
soil by bioaugmentaUon. Appl Microbiol Biotechnol 1993,
38:681-687.
29. RomichMS, Cameron DC, Etzel MR: Three methods for
large-scale preservation of a microbial inoculum for
bioremedistion. In Bioaugmentation for Site Remediation. Edited
by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle
Press; 1995:229-235.
30. Liang R, McFadand MJ: Biodegradation of pentachlorophenol
in soil amended with the white rot fungus Phanerochaete
chrysosporium. Hazard Waste Hazard Mater 1994, 11:411-421.
31. Petrich CR, Stormo KE, Knaebel DB, Ralston DR, Crawford RL: A
preliminary assessment of field transport experiments using
encapsulated cells. In Bioaugmentation for Site Remediation.
Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus:
Battelle Press; 1995:237-244.
32. Bajpai RK, Zappi ME, Gunnison D: Additives for establishment
of biologically active zones during in situ bioremedlation. Ann
NY Acad Sci 1994, 721:450-465.
33. Selenska-Pobell S: How to monitor released rhizobia. Plant Soft
1994, 166:187-191.
34. Burlage RS, Palumbo AV, McCarthy J: Signal quantification
of bacteria for a groundwater transport experiment. In
Bioaugmentation for Site Remediation. Edited by Hinchee RE,
Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:139-148.
35. Masson L, Comeau Y, Brousseau R, Samson R, Greet C:
Construction and application of chromosomally integrated
lac-lux gene markers to monitor the fate of a 2,4-
dichlorophenoxyacetlc acid-degrading bacterium in
contaminated soils. Microb Releases 1993, 1:209-216.
36. Blackburn NT, Seech AG, Trevors JT: Survival and transport
• of lac-lux marked Pseudomonas fluorescens strain in
uncontaminated and chemically contaminated soils. Syst Appl
Microbiol 1994, 17:574-580.
Reports an interesting use of luminescent bacteria for monitoring bacterial
transport and survival. Bacteria are shown to survive better at lower temper-
atures (10"C) and in non-contaminated soils. The influence of contaminated
soils on the survival of bacteria capable of growing on the contaminant
(PAHs) is not shown.
3?. King JMH, Digrazia PM, Applegate B, Burlage R, Sanseverino J,
Dunbar P, Larimer F, Sayler GS: Rapid, sensitive bioluminescent
reporter technology for naphthalene exposure and
biodegrsdation. Science 1990, 249:778-781.
38. Venosa AD, Suidan MT, Haines JR, Wrenn BA, Strohmeier KL,
Eberhart BL, Kadkhodayan M, Holder E, King D, Anderson B:
Field bioremediation study: spilled crude oil on Fowler Beach,
Delaware. In Bioaugmentation for Site Remediation. Edited by
Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:49-56.
39. Dybas MJ, Tatara GM, Knoll WH, Mayotte TJ, Criddle CS: Niche
adjustment for bioaugmentation with Pseudomonas sp.
strain KC. In Bioaugmentation for Site Remediation. Edited by
Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:77-84.
40. Witt ME, Dybas MJ, Heine RL, Nair S, Criddle CS, Wigged DC:
Bioaugmentation and transformation of carbon tetrachloride in
a model aquifer. In Bioaugmentation for Site Remediation. Edited
by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle
Press; 1995:221-227.
41. Holroyd ML, Caunt P: Large-scale soil bioremediation using
white-rot fungi. In Bioaugmentation for Site Remediation. Edited
by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle
Press; 1995:181-187.
42. Edgehill RU: Removal of pentachlorophenol from soil by
Arthrobacter strain ATCC 33790. In Bioaugmentation/or Site
Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC.
Columbus: Battelle Press; 1995:85-90.
43. Shonnard DR, Taylor RT, Hanna ML, Boro CO, Duba AG:
• Injection-attachment of Methylosinus trichosporium OB3b in a
two-dimensional minature sand-filled aquifer simulator. Water
Resource Res 1g94, 30:25-35.
Description of the possible factors influencing the attachment of injected
bacteria in sandy aquifers. The experiments did not study cell straining, but
did investigate colloid-like filtration, where solution ionic strength plays an
important role.
44. JenningsDA, Petersen JN, Skeen RS, Peyton BM, Hooker
BS, Johnstone BL, Yonge DR: An experimental study of
316 Environmental biotachnology
microbial transport in porous media. In Bioaugmentation for Site
Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC.
Columbus: Battelle Press; 1995:97-103.
45. Bryers JD, Sanin S: Resuscitation of starved ultramicrobacteria
to improve in situ bloremediation. Ann NY Acad Sci 1994,
30:61-76.
46. Ramos-Cormenzana A, Castillo A, Incerti C, Gomez-Palma LF:
Bacteriological indicators of faecal contamination: result of a
loading experiment with untreated urban wast°water. J Appl
Bacteriol 1994, 76:95-99.
47. Kim SH, Corapcioglu MY: Effects of mobile bacteria in
bloremediaUon operations. In Bioaugmentation for Site
Remediation. Edited by Hinchee RE, Fredrickson J, All°man BC.
Columbus: Battelle Press; 1995:91-96.
48. Vent°tea RT, Hicks RJ, Lewis RF: Comparison of three lignin-
degrading fungi for degrading cyclodiene insecticides. In
Bioaugmentation for Site Remediation. Edited by Hinchee RE,
Fredrickson J, All°man BC. Columbus: Battelle Press;
1995:157-164.
49. Field JA, Feiken H, Hag° A, Kotterman MJJ: Application
of a white-rot fungus to biodograde benzo(a)pyrene in
soil. In Bioaugmentation for Site Remediation. Edited by
Hinchee RE, Fredrickson J, All°man BC. Columbus: Battelle Press;
1995:165-171,
50. Kotterman MJJ, Wasseveld R, Field JA: Influence of nitrogen
sufficiency and manganese deficiency on PAH degradation by
Bjerkandera sp. In Bioaugmentation for Site Remediation. Edited
by Hinchee RE, Fredrickson J, All°man BC. Columbus: Battelle
Press; 1995:189-194.
51. Lin J-E, Lantz S, Schultz WW, Mueller JG, Pritchard PH: Use of
microbial encapsulation/immobllizaton for biodegradaUon
of PAHs. In: Bioaugmentation for Site Remediation. Edited by
Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press;
1995:211-220.
52. Leung K, Cassidy MB, Holmes SB, Lee H, Trevors JT: Survival
of k-carrageenan-encapsulated and unencapsulatad
Pseudomones aeruginosa UG2Lr cells In forest soil monitored
by polymerese chain reaction and spread plating. FEMS
Microbio/Ecol 1995, 16:71-82.
53. Vandepitte V, Quataert P, De Rore H, Verstraete W: Evaluation of
•* the Gompertz function to model survival of bacteria Introduced
into soils. Soil Biol Biochem 1995, 27:365-3'72.
Describes the best function, to date, for describing the asymptotic changes
in added microbial populations, application of which could aid in accurately
determining the effective concentrations of microorganisms for bioaugmen-
ration. Significantly, the fitting of this function often provided results that
were logical (e.g.E. coil had final populations considerably smaller than
Pseudomonas sp.)
54. Schloter M, Abmus B, Hartmann A: The use of immunological
• methods to detect and identify bacteria in the environment.
Biotechnol Adv 1995, 13:75-90.
A general review of the potential uses and problems of immunological meth-
ods for monitoring bacteria in environmental samples.
55. Winkler J, Timmis KN, Snyder RA: Tracking the response of
• Burkholderia cepacia G4 5223-PR1 in aquifer microcosms.
Appl Environ Microbiol 1995, 61:448-455.
Describes an interesting application of monoclonal antibody immuno-
fluorescence for counting added microorganisms under different experimen-
tal conditions. The technique was able to compare the effect of sterile non-
sterile conditions on bacterial survival with microorganisms that lacked niche
adaptation.
56. Barkay T, Kroer N, Rasmussen LD, S6rensen SJ: Conjugal
transfer at natural population densities in a microcosm
simulating an estuarlne environment. FEMS Microbiol Ecol
1995, 16:43-54.
5?. Neilson JW, Josephson KL, Pepper IL, Arnold RB, Di Giovanni GD,
Sinclair NA: Frequency of horizontal gene transfer of a large
catabolic plasmid (pJP4) in soil. Appl Environ Microbiol 1994,
60:4053-4058.
58. Wsckett LP, Lange CC, Ornstein RL: BiodehalogenaUon:
natural and engineered systems. In Bioaugmentation for Site
Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC.
Columbus: Battelle Press; 1994:105-114.
59. Heuer H, Dwyer DF, Timmis KN, Wagner-D6bler h Efficacy in
aquatic microcosms of a genetically engineered pseudomonad
applicable for bioremediatlon. Microb Ecol 1995, 29:203-220.
60. Anderson TA, Coats JR (Eds): Blot°mediation Through Rhiosphere
Technology. ACS Symposium Series 563. Washington, DC:
American Chemical Society; 1994.