Microbial Cell Factories
BioMed Central
Open Access
Review
A review on slurry bioreactors for bioremediation of soils and
sediments
Ireri V Robles-González1, Fabio Fava2 and Héctor M Poggi-Varaldo*1
Address: 1CINVESTAV-IPN, Environmental Biotechnology R&D Group, Dept. Biotechnology and Bioengineering, México D.F., México and 2Alma
Mater Studiorum-University of Bologna; Faculty of Engineering, Viale Risorgimento, 2. 40136. Bologna, Italy
Email: Ireri V Robles-González - irerirobles@yahoo.com.mx; Fabio Fava - fabio.fava@unibo.it; Héctor M PoggiVaraldo* - hectorpoggi2001@gmail.com
* Corresponding author
Published: 29 February 2008
Microbial Cell Factories 2008, 7:5
doi:10.1186/1475-2859-7-5
Received: 7 November 2007
Accepted: 29 February 2008
This article is available from: http://www.microbialcellfactories.com/content/7/1/5
© 2008 Robles-González et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The aim of this work is to present a critical review on slurry bioreactors (SB) and their application to
bioremediation of soils and sediments polluted with recalcitrant and toxic compounds. The scope of the review
encompasses the following subjects: (i) process fundamentals of SB and analysis of advantages and disadvantages;
(ii) the most recent applications of SB to laboratory scale and commercial scale soil bioremediation, with a focus
on pesticides, explosives, polynuclear aromatic hydrocarbons, and chlorinated organic pollutants; (iii) trends on
the use of surfactants to improve availability of contaminants and supplementation with degradable carbon
sources to enhance cometabolism of pollutants; (iv) recent findings on the utilization of electron acceptors other
than oxygen; (v) bioaugmentation and advances made on characterization of microbial communities of SB; (vi)
developments on ecotoxicity assays aimed at evaluating bioremediation efficiency of the process.
From this review it can be concluded that SB is an effective ad situ and ex situ technology that can be used for
bioremediation of problematic sites, such as those characterized by soils with high contents of clay and organic
matter, by pollutants that are recalcitrant, toxic, and display hysteretic behavior, or when bioremediation should
be accomplished in short times under the pressure and monitoring of environmental agencies and regulators. SB
technology allows for the convenient manipulation and control of several environmental parameters that could
lead to enhanced and faster treatment of polluted soils: nutrient N, P and organic carbon source (biostimulation),
inocula (bioaugmentation), increased availability of pollutants by use of surfactants or inducing biosurfactant
production inside the SB, etc. An interesting emerging area is the use of SB with simultaneous electron acceptors,
which has demonstrated its usefulness for the bioremediation of soils polluted with hydrocarbons and some
organochlorinated compounds. Characterization studies of microbial communities of SB are still in the early
stages, in spite of their significance for improving reactor operation and design optimization.
We have identified the following niches of research needs for SB in the near and mid term future, inter alia: (i)
application of SB with sequential and simultaneous electron acceptors to soils polluted with contaminants other
than hydrocarbons (i.e., pesticides, explosives, etc.), (ii) evaluation of the technical feasibility of triphasic SB that
use innocuous solvents to help desorbing pollutants strongly attached to soils, and in turn, to enhance their
biodegradation, (iii) gaining deeper insight of microbial communities present in SB with the intensified application
of molecular biology tools such as PCR-DGGE, PCR-TGGE, ARDRA, etc., (iv) development of more
representative ecotoxicological assays to better assess the effectiveness of a given bioremediation process.
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1. Introduction
Bioremediation is an alternative that offers the possibility
to destroy toxic pollutant using natural biological activity.
By definition, bioremediation is the use of living organisms, primarily microorganisms, to degrade the environmental contaminants into less toxic forms. It uses
naturally occurring bacteria and fungi or plants to degrade
or detoxify substances hazardous to human health and/or
the environment [1]. The microorganisms may be indigenous to a contaminated area or they may be isolated from
elsewhere and brought to the contaminated site. Contaminant compounds are transformed by living organisms
through reactions that take place as a part of their metabolic processes. Biodegradation of a compound is often a
result of the actions of multiple organisms. When microorganisms are imported to a contaminated site to enhance
degradation we have a process known as bioaugmentation [2]. Bioremediation techniques are typically more
economical than thermal and physico-chemical remediation such as incineration [3,4].
Bioremediation processes have been classified in three
broad categories, according to place and soil handling/
conditioning: in situ, ad situ, and ex situ. The second and
third class of processes are useful for the remediation of
(1) sludges, soils or sediments polluted with high concentration of recalcitrant contaminants [5], for instance polynuclear aromatic hydrocarbons [6-9], diesel [10,11],
explosives [12], pesticides and chlorinated organic pollutants [13,14], oily sludges from the petrochemical industry
[15]; (2) clayish and stratified soils with low hydraulic
conductivity and low permeability accompanied with
high contents of organic matter [13,16]; and (3) soils in
regions and areas where environmental conditions are
adverse to biological processes, for instance, low temperature that negatively affects biodegradation rates [17], and
(4) contaminated sites that require a short remediation
time because of regulatory or other pressures [18-20].
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diation of polluted soils and sediments. The scope of the
review encompasses the following subjects: (i) process
fundamentals of SB and analysis of advantages and disadvantages; (ii) the most recent applications of SB to laboratory scale and commercial scale soil bioremediation, with
a focus on pesticides, explosives, polynuclear aromatic
hydrocarbons, and chlorinated organic pollutants; (iii)
trends on the use of surfactants to improve bioavailability
of contaminants and supplementation with biodegradable carbon sources to enhance cometabolism of pollutants; (iv) recent findings on the utilization of electron
acceptors other than oxygen; (v) advances made in characterization of microbial communities of SB; (vi) developments on ecotoxicity assays aimed at evaluating
bioremediation efficiency of the process.
2. Process fundamentals and analysis of
advantages and disadvantages of slurry
bioreactors
Slurry bioreactor technology is an engineered complex
that generally comprises four parts: installations for polluted soil handling and conditioning, the bioreactor battery itself, installations for treated soil handling and
disposal, and ancillary equipment for treatment of process by-streams [18,20]. The SB can be classified as batch,
semi-continuous, and continuous from the operation
point of view. The most common operational mode is
batch. Another useful classification relies on the main
electron acceptor used in the biodegradation process: aerobic (molecular oxygen), anoxic (nitrate and some metal
cations), anaerobic (sulfate-reducing, methanogenic, fermentation) [13], and mixed or combined electron acceptors [29-31]. By far, aerobic SBs have predominated in full
scale applications, although anaerobic SBs are an emerging area of research and development [21].
Slurry bioreactors are one of the most important types of
ad situ and ex situ technique. Treatment of soils and sediments in slurry bioreactors has become one of the best
options for the bioremediation of soils polluted by recalcitrant pollutants under controlled environmental conditions [17,21,22]. SBs are also very often applied to
determine the feasibility and actual potential of a biological strategy in the final restoration of a contaminated soil
or site [8,11,14]. In fact, under slurry conditions, the pollutant depletion rates depend mainly on the degradation
activity of the microorganisms available in the system [20]
and the results obtained generally reflect the actual biological depuration potential of the soil [23-28].
Interesting and distinctive features of SBs are that soil is
treated in aqueous suspension, typically 10 to 30% w/v
and that mechanical or pneumatic mixing is provided.
These characteristics, in turn, lead to several process
advantages, inter alia: (i) increased mass transfer rates and
increased contact microorganisms/pollutant/nutrients;
(ii) increased rates of pollutant biodegradation compared
to in situ bioremediation or to ad situ solid phase biotreatment: (iii) associated to (i) and (ii), significantly shorter
treatment times can be achieved; (iv) possibility of using
different electron acceptors (O2, SO4-2, CO2, NO3-); (v)
control and optimization of several environmental
parameters such as temperature, pH, etc.; (vi) effective use
of biostimulation and bioaugmentaion; (vii) increase pollutant desorption and availability through the addition of
surfactants and solvents [2,10,12,13,32].
The objective of this paper is to present a critical review on
slurry bioreactors (SBs) and their application to bioreme-
However, SBs also have a few disadvantages, all of them
related to requirements for soil excavation, handling, and
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conditioning, and bioreactor construction/operation that
typically increase treatment costs compared to most simple bioremediation techniques [20]. In spite of this, SBs
has resulted more cost effective than incineration, solvent
extraction and thermal desorption in many cases [33].
2.1 Process description
In SB, soil is excavated and conditioned and loaded into
bioreactors [17,34]. A main feature of SB is that soil inside
reactor is kept in aqueous suspension by some type of
mixing in a way that biological treatment is carried out
under saturated conditions [18] and nearly homogeneous
suspension [17] (See Figure 1).
There are several bioreactor configurations [20,21]. At full
scale, low cost bioreactors may consist of large lined
lagoons (24 m × 15 m). Manufactured bioreactors can
range 3 to 25 m diameter and 4.5 to 8 m height, with
capacities between 60 to 1000 m3 [12,18,19]. Bioreactors
are usually fitted with mixing devices, and aerobic SB are
equipped with spargers or diffusers. Ancillary equipment
may include gas emissions conduits and treatment, vessels for nutrient and pH conditioning of slurry, etc.
Main operating modes of SB include batch and semi-continuous. Continuous SB are possible in principle, but are
not very common. Batch and semi-continuous reactors
adapt easily to the handling of soils and slurries [18,35],
sometimes are operated in sequencing batch reactors
[5,20,21].
2.2 Soil pretreatment
As it was mentioned above, one of the limitations of SBs
technology is the requirement of soil excavation and pretreatment. Usually, pretreatment consists of crushing followed by screening. The coarser fractions of soils (pebbles
and sands, 0.85 to 4 mm) are discarded and sent to direct
disposal, whereas fine fractions (clay and organic matter,
< 0.85 mm) are retained and loaded into bioreactors. It is
generally recognized that pollutants concentrate in fine
particles of soil [16,19].
2.3 Solids concentration
Polluted, fine fractions of soils are mixed with water or
wastewater to form slurry with a concentration in the
range between 15 to 60% w/v, depending on characteristics of soil and degradation rates determined in previous
laboratory or pilot scale studies [20,21]. Solids concentration is a key variable that might determine the mixing
power required, aeration efficiency in aerobic SB, and the
size of by-stream post-treatment installations [18]. It is a
common practice to use decanted spent liquors from pre-
Treatment of
exhaust gas
Treated gas
Make-up
water
Recycled water
POLLUTED SOIL
Wastewater
treatment
Slurry Bioreactor
Clarifier
Addition of nutrients,
inoculum,
surfactants
and electron acceptors
Excess of water
TREATED SOIL
Figure
Flow
diagram
1
of a typical slurry bioreactor installation
Flow diagram of a typical slurry bioreactor installation. Clarifier is optional.
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vious batch runs as water for making the slurries, as a
method to minimize liquor treatment and disposal.
2.4 Mixing
SBs are provided with mixing devices in order to keep solids in suspension during biotreatment, as well as intensifying turbulence and thus increasing mass transfer rates
[21]. There are several types of mixing devices, mechanical
and pneumatic ones are the predominant among aerobic
SBs. Also, mixing can be intermittent or continuous. The
first leads to significant savings on power expenses,
although mixing intensity is lower.
Mixing intensity is a critical factor in SB design and performance [21]. Its main role is to keep solid particles in
suspension and provide slurry homogeneization, to help
achieving a satisfactory aeration in aerobic SBs, and to
increase several mass transfer rates such as desorption of
pollutants from soil, among others. This, in turn, usually
translates in higher rates of contaminant biodegradation,
in particular when difficult-to-degrade xenobiotic compounds such as PCBs have to be removed [14]. Mixing
selection and sizing mainly depends on slurry characteristics and kinetic requirements. Pilot studies and semiempirical methods are usually required. Independently of
the type of mixer selected, mixing power is of major concern because of its influence on the operating costs. The
denser the slurry, the higher the power and more difficult
to achieve oxygen transfer in aerobic SBs [19,34]. So, several trade-offs have been recommended to keep power
expenses at bay, such as intermittent mixing and slurry
dilution [18].
2.5 Control of environmental conditions in the operation
of slurry bioreactors
One distinctive and advantageous feature of SBs is the
manipulation and control of environmental conditions
that leads to biodegradation optimization and better
process performance [17,21,35]. In this regard, several
operational variables can be monitored and controlled:
pH, dissolved oxygen in aerobic SBs, concentration of
inorganic nutrients, pH is usually kept in the range
6.75–7.25 using either alkalis such as NaOH or acids such
as H2SO4. Temperature range is 25–30°C, although SBs
have been reported to successfully perform at lower ambient temperatures. Ideal dissolved oxygen concentration is
90% of the saturated dissolved concentration. Nitrogen
and phosphorus salts are used as inorganic nutrient
sources in order to ensure that there is no nutrient limitation (NH4Cl, KH2PO4, Na2HPO4) [5,17,36].
3. Applications of aerobic slurry bioreactors to
bioremediation of soils polluted with pesticides,
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organo-chlorinated substances, explosives, and
polynuclear aromatic hydrocarbons
Application of aerobic SBs (A-SB) to bioremediation of
soils is still predominant. Aerobic degradation of organic
pollutants is carried out by its oxidation via oxygenases or
dioxygenases, so, molecular oxygen is usually a limiting
factor [37]. A large number of laboratory, pilot and full
scale studies and cases of A-SB can be found in the literature. In Tables 1, 2 and 3 we have excerpted some applications of A-SB to bioremediation of soils polluted with
PAHs, pesticides and PCBs, and explosives, respectively.
Jee et al. [38] studied the bioremediation of sediment contaminated by 88 mg/kg phenanthrene. The influence of
different solid/sediment loads and mixing conditions on
the time course of the process was investigated. Under
well mixed conditions, phenanthrene was removed by 25
to 40% during the first few days of treatment by showing
an activity which decreased markedly from the 3rd to the
5th day. In unmixed systems, the extent of mineralization
was notably lower, ranging from 5 to 20%. The pollutant
mineralization did not cease by end of the 7th day of treatment. Under well mixed conditions, the reactors with a
10% sediment load showed the highest level mineralization. Pollutant mineralization yields achieved in the reactors with 5 and 15% sediment were similar, and lower
than those attained with 10% sediments. In the unmixed
reactors, the 10 and 15% solids loaded reactors showed
similar activity.
Dean-Ross [39] conducted a A-SB bioremediation batch
experiment on a pristine sediment spiked with phenanthrene, anthracene, pyrene and fluoranthene. PAHs, each
added at 60–70 mg/L, disappeared after 6 to 8 days of
treatment. Bioaugmentation with Rhodococcus sp. resulted
in enhanced bioremediation rates and yields.
Villermur et al. [40] used a three 3-phase (two liquid
phases + soil, TLP) aerobic slurry system containing silicone oil at 30% v/v to biodegrade selected high molecular
weight PAHs spiked into a soil at 196 mg pyrene/kg, 192
mg chrysene/kg, and 163 mg benzo [a]pyrene/kg. Pyrene
was degrade d through a rate of 19 mg/(L day) whereas the
degradation rates of chrysene and benzo [a]pyrene were,
3.5 and 0.94 mg/(L day), respectively.
Wang & Vipulanandam [9] observed the effect of
naphtalene concentration on the A-SB remediation potential. Naphthalene was generally found to rapidly desorb
from the spiked soil by undergoing rapid and extensive
biodegradation. 500 mg/kg of naphthalene were reduced
to 20 mg/kg after 65 h days of treatment whereas 5000
mg/kg of the same pollutants were reduced to 40 mg/kg
after 100 h.
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Table 1: Remediation of sites/actual site soils contaminated with PAH in aerobic slurry bioreactors.
Matrix
Contaminant
Remarks
Removal
Ref.
Soil
PAHa:
Pyrene
PAHsa
a) without surfactant
b) Brij 30
- Corynebacterium aquaticum
- Flavobacterium mizutaii
- Mycobacterium gastri
- Pseudomonas aeruginosa
- Pseudomonas putida
- Biosurfactant
- PAH-degrading consortium
- 30% (v/v) silicone oil
a) 63%
b) 70%
93%
[106]
a) 19 mg L-1 day-1
b) 3.5 mg L-1 day-1
c) 0.94 mg L-1 day-1
[40]
a) Clay soil
b) Sand soil
a) Mixed system
b) Unmixed system
- 7 days incubation
- 5, 10, 15% sediment load
Pseudomonas putida
a) 43%
b) 25%
a) 25 – 40%
b) 5 – 20%
[41]
100%
[107]
Petroleum waste sludges
90%
[33]
Flavobacterium sp.
a) 96%
b) 99%
c) 99%
[9]
Slurry-phase in the absence and
presence of soya lecithin or humic
substances at 1.5% w/w
Comparison SB, blade agitated
bioreactor and rotary vessel
bioreactor
Absidia cylindrospora Maltosylcyclodextrin
Rhodococcus sp.
25% (without agents) 58% (with
agents)
[11]
SB was capable of the readiest and
fastest removal of soil PAHs
[8]
Soil
Soil
Sandy
Soil
PAHsa:
a) Pyrene (358 mg/kg),
b) chrysene (255 mg/kg),
c) benzo [a]pyrene (250 mg/kg)
PAHsa
Estuarine sediments
PAHa
Phenanthrene
Soil
PAHa
Naphthalene
PAHsa
Pyrene, benzo [a]anthracene,
chrysene
PAHa
a) Naphthalene (500 mg/kg)
b) Naphthalene (5000 mg/kg)
c) Naphthalene (25000 mg/kg)
Complex mixture of PAHsa(~13.0
g/kg)
Waste sludges
Soil
Clay 20%
Sand 79%
Organic matter 1%
Actual site soil
Actual site soil
Complex mixture of PAHsa (~3.7 g/
kg)
Soil
PAHa Fluorene
a) Pristine sediment
Clay 9%
Silt 16%
Sand 75%
Organic matter 1.7%
b)Contaminated sediment
Clay 6%
Silt 5%
Sand 89%
Organic matter 5.1%
a) supplemented with PAHsa:
phenanthrene, anthracene, pyrene
and fluoranthene (60–70 ppm)
b) PAHsa:
phenanthrene (36.5 ppm)
anthracene (110.2 ppm)
pyrene (350 ppm)
fluoranthene (150 ppm)
[92]
[38]
90%
[108]
a) 100%
b) >98%
[39]
Notes: aPAHs: Polynuclear aromatic hydrocarbons
Castaldi [33] treated petroleum waste sludges containing
680 mg/Kg of 2–3 ring PAHs and 38 mg/kg of 4–6 ring
PAHs in a continuous flow multistage A-SB that operated
at relatively short residence times with minimal loss of
volatiles constituents. The higher molecular weight PAHs,
including the four-ring derivatives of pyrene, benzo
[a]pyrene, and chrysene, were removed with efficiencies
greater than 90%.
Rutherford et al. [41] treated in a A-SB two different soils
(a sandy soil and a silty clay one) contaminated with creosote compounds in the range 10 000 to 20 000 mg/kg.
Unexpectedly, the extent of PAHs removal over 10 weeks
was higher in the clay soil (43%) than in the sandy one
(25%).
De Jonge & Verstrate [42] reported 70% removal from a
soil initially polluted with up to 3 000 mg/kg of oil in ASBs. Also, soils heavily contaminated with crude oil (200
g TPH/kg) could be treated in pilot A-SBs reaching 1 to 2
g TPH/kg in 5–7 weeks of treatment [43].
Castaldi & Ford [15] reported 73% removal of PCBs in 90
days of A-SB treatment of petrochemical waste sludges
polluted with 115 mg/kg of PCBs. Bioremediation of sediments contaminated with 88 mg phenanthrene/kg in
batch A-SB showed removal percentages of 25–50% in 7
days of operation [36]. On the other hand, Mueller et al.
[17] observed the extensive removal (up 98%) of pentachlorophenols and creosote from soils and sediments in
30 days of operation. A soil spiked with a mixture of PAHs
(270 mg/kg), composed by fluorene, phenanthrene,
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Table 2: Remediation of sites/actual site soils contaminated with pesticides and PCBs in aerobic slurry bioreactors.
Matrix
Contaminant
Remarks
Removal
Ref.
Waste activated sludge
EPTCa (5 – 6 ppm)
γ-HCHb
a) freely suspended activated sludge
b) immobilized activated sludge
Inoculated with Pseudomonas putida
coexpressing cytocrome P-450 cam and
luciferase
a)35%
b)72%
65%
[109]
Agricultural soil
Clay 56.11%
Silt 23.2%
Sand 20.69%
pH 8.3
Organic carbon 0.6%
Soil
Clay 13%
Silt 21%
Sand 66%
pH 7.7
Total carbonates 3%
Actual site contaminated soil
a) PCBsc (1210 mg/kg)
b) TPHd (11407 mg/kg)
Rhamnolipid production was observed by
Pseudomonas aeruginosa
a) 98%
b) 99%
[49]
PCBsc (1547 mg/kg)
Inoculated with ECO3 co-culture
[47]
Actual site contaminated Soil
PCBsc (1547 mg/kg)
[14]
Actual site contaminated Soil
PCBsc (350 mg/kg)
Soil
Clay 48%
Silt 41%
Sand 11%
pH 7.0
Organic matter 4%
Soil
Clay 26%
Silt 48%
Sand 26%
pH 7.2
Organic matter 0.8%
2,4-De (300 mg/kg)
Inoculated with ECO3 co-culture;
optimization of SR configuration
Inoculated with ECO3 co-culture in the
absence and the presence of
Cyclodextrins, Quillaya Saponin, Triton
X-100
With and without sucrose
21% (without inoculum)
39% (with inoculum)
18% (in shake flasks with baffles)
30% (in stirred thank reactor)
65% (without agents) 80% (with agents)
>90%
[30]
a) 47.9%
b) 75.4%
[22]
DEPf (8 mg/g)
a) with native soil microflora
b) with effluent treatment plant
microflora
[48]
[46]
Notes: aS-ethyl dipropylthiocarbamate; bγ-hexaclhorocyclohexane; cPolychlrorinated byphenyls; dTotal petroleum hydrocarbons; e2,4dichlorophenoxyacetic acid; fdi-ethyl phthalate
anthracene and pyrene, was treated in a batch A-SB for 20
days: fluorene and phenathrene were found to be
removed by 70% and 40% after 3 days of incubation [16].
In another work with soil polluted with PAHs, Lewis [44]
reported a removal of 70–97% of spiked PAHs (728 – 4
920 mg/kg) in 60 days of treatment.
Pinelli et al. [8] studied the intrinsic depuration capability
of a soil historically contaminated by PAHs (overall initial
PAHs: 3.7 g/kg) by using different aerobic batch bioreactors: a SB, a blade agitated bioreactor and a rotary vessel
bioreactor. The performance of each bioreactor was evaluated by analyzing the disappearance of 14 target PAHs
Table 3: Remediation of sites/actual site contaminated with explosives in aerobic slurry bioreactors.
Matrix
Contaminant
Remarks
Removal
Ref.
Soil from Joliet Army Ammunition Plant
2,4,6-TNT a
2,4,6-TNBb
a) 2,4-DNT b (14715 mg/kg) 2,6-DNTc (8940
mg/kg)
b) 2,4-DNT b (1125 mg/kg) 2,6-DNTc (4800
mg/kg)
Addition of molasses
90%
[12]
Augmentation with a DNTmineralizing culture
98%
[5]
2,4,6 trinitrophenylmethylnitramined
2,4,6-TNTa
-
99%
40%
[110]
[111]
Soils from Army Ammunition Plant
a) soil VAAP
moisture 3%
bulk density 1.4 g/ml
pH 4.6
b) soil BAAP
moisture 1.6%
bulk density 1.7 g/ml
pH 9,35
Soil
Soil
Notas: a2,4,6-trinitrotoluene; b2,4,6-trinitrobenzene
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and of the total extractable organic matter of the soil.
About 72, 96 and 95% of the total amount of PAHs initially present in the soil were removed in the SB, blade agitated semisolid-phase bioreactor, and rotatory vessel
semisolid-phase bioreactor after 17, 47, and 35 days of
treatment, respectively. Among the three treatments, SB
was capable of inducing the readiest and fastest removal
of the soil PAHs.
Fava et al. [11] treated an actual site historically contaminated soil containing about 13 g/kg of a large variety of
PAHs in laboratory scale A-SB amended with soya lecithin
or humic substances at 1.5% w/w. After 150 days of incubation of a room temperature, about 60% of original
amount of PAHs was biodegraded with a marked reduction of the original soil ecotoxicity.
A-SBs have also been successfully applied to bioremediation of soils contaminated with pesticides. Cookson [20]
reported that levels of pesticides in soils polluted with
mixtures of 2,-4-dichlorophenoxy acetic acid (2,4-D), 4chloro-2-methyl-phenoxy acetic acid, alachlor, trifuralin
and carbofuran could be reduced from 800 to 20 mg/kg
after two weeks of treatment. Robles-González et al. [30]
carried out laboratory scale experiments with batch A-SB
for bioremediation of an agricultural soil with high contents of clay and organic matter and polluted with 300 mg
2,4-D/kg. They observed removals of 2,4-D higher than
95% after 14 days of treatment; no chlorophenol intermediates of 2,4-D degradation were detected. Bachman et al.
[27] reported 90–100% mineralization of α-hexachlorocyclohexane (HCH) from a soil initially polluted with 400
mg α-HCH/kg after 42 days of batch incubation of laboratory scale A-SB.
SBs also provided excellent results in the aerobic bioremediation of actual site PCBs-contaminated soils. Fava and
co-workers [45,46] reported of the efficient bioremediation of an actual site soil with 350 mg/kg of total PCBs
through 141 days of A-SB treatment. They also demonstrate the possibility of enhancing the process through the
SB supplementation with cyclodextrins (CDs) or the phytogenic surfactant Quillaya Saponin at 10 g/L. The opportunity
to
improve
the
aerobic
slurry-phase
bioremediation of another actual site soil lacking indigenous specialized bacteria and contaminated by 1547 mg/
kg of PCBs through (a) soil bioaugmentation with the
PCB mineralizing three-member bacterial coculture ECO3
[47] or (b) improvement of SB configuration [14] was
successively investigated and demonstrated by the same
authors.
Rattan et al. [48] found that batch soil slurry microcosms
inoculated with a strain of Pseudomonas putida coexpressing cytochrome p-450 cam and luciferase, removed more
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than 65% of γ-HCH (105 mg/L slurry) after 4 weeks of
incubation. On the other hand, less than 15% was the γHCH depletion achieved in the parallel non-inoculated
controls.
Hudak & Cassidy [49] treated an aged contaminated soil
in SB; lubricating oil (LO) and polychlorinated biphenyls
(PCBs) were the pollutants applied at the concentration of
~11 400 mg/kg and ~1200 mg/kg, respectively. They also
examined biosurfactant production by Pseudomonas aeruginosa. Rhamnolipid production was observed within 1 to
2 days after nitrogen depletion. By day 6, total rhamnolipid concentrations increased from below detection to
average values over 1 000 mg/L, which was associated to
removals over 98% of soil-bound PCBs and over 99% of
total petroleum hydrocarbons.
Mohan et al. [22] studied the degradation of a di-ethyl
phthalate (DEP) contaminated soil in a sequencing A-SB;
the effect of bioaugmentation using effluent treatment
plant (ETP) microflora on the degradation of DEP was
also investigated. Consistent desorption of DEP was
observed in the aqueous phase with increase in the contact time (cycle period) due to prevailing agitation in the
reactor. Reactor operated with native soil microflora (7.6
× 103 CFU/g) and exhibited a maximum DEP degradation
of 47.9%. Initially, up to 8 h of the cycle operation, the
degradation of DEP in soil was found to be 9.85% and
reached a maximum of 47.9% in 40 h. Another reactor
was operated in the presence of ETP-degrading microflora
(2.4 × 107 CFU/ml) without native soil microflora. Within
8 h of cycle operation a DEP degradation of 22.45% was
noticed and reached a maximum of 75.4% at the end of
cycle period. It was concluded that bioaugmentation with
ETP-acclimated biomass significantly increased DEP
removal.
Zhang et al. [5] have conducted pilot scale studies on
bioremediation with A-SB of soils contaminated with
explosives. They treated two soils from a former ammunition manufacturing plant, polluted with 14715 mg/kg to
1125 mg/kg of 2,4-dinitrotoluene (2,4-DNT) and 8940
mg/kg to 4800 mg/kg of 2,6-dinitrotoluene (2,6-DNT).
Almost complete removal of explosives could be achieved
in a short 2 days period. Moderate removal of 40% was
reported for a soil polluted with 570 mg/kg of 2,4-DNT
and 380 mg/kg of 2,6-DNT [50]. Laboratory scale experiments with A-SB showed an effective removal of explosives (90%) from soils with initial 400–1200 mg TNT/kg
after 130 days of treatment [12].
4. Utilization of electron acceptors other than
oxygen
Recent research has shown that non aerobic bioremediation holds promise for remediation of specific recalcitrant
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and toxic contaminants (such as organo-chlorinated pesticides and compounds, nitro-organics, etc.) where soils
are rich in sulfate, nitrate, or bicarbonate [13,30,51-54].
Very often, non aerobic bioremediation is also preferable
when anaerobic or reducing conditions predominate in
the contaminated sites. In such scenarios, anaerobic
bioremediation may be desirable whenever the pollutants
are amenable to reductive dechlorination or nitro-reduction as first step to final biodegradation. Another advantage of anaerobic remediation is associated to savings of
aeration (both investment and operating costs of aeration) [29,30].
Reported studies on anaerobic bioremediation of soils
and/or sediments in SB are still scarce. Table 4 summarizes a selection of recent research published in the open
literature. Boopathy [29] treated a soil contaminated with
550 mg diesel/kg in several anoxic and anaerobic batch,
laboratory scale SB. He found the highest removal of 80%
in a SB with "mixed" or combined electron acceptors
(SO4=, NO3-, CO2). One electron acceptor-SB showed
lower removals, for instance, 55% of diesel removal was
observed in the sulfate-reducing SB. Soils polluted with
1500 mg TNT/kg have been treated in batch, pilot scale
anaerobic SB; up to 95% removal of TNT was reported
[55].
Bachman et al. [28] tested aerobic and several laboratory
scale anaerobic SB for the bioremediation of a coarse soil
polluted with 400 mg α-HCH/kg. They reported 90 to
100% mineralization of the pesticide after 100 days of
treatment in the A-SB, 85% in the methanogenic SB,
whereas in the sulfate-reducing and denitrifying SB no
conversion of HCH was observed. A 95% removal of
Dinoseb (2-sec-buthyl-2,6-dinitrophenol) in methanogenic conditions was obtained from a soil contaminated
with 800 mg/kg of this herbicide [55]. Robles-González et
al. [30] conducted laboratory scale experiments with
batch sulfate-SB for bioremediation of an agricultural soil
with high contents of clay and organic matter and polluted with 300 mg 2,4-D/kg. They observed removals of
2,4-D near to 50% after 30 days of treatment and they
detected chlorophenol intermediates of the anaerobic
transformation of 2,4-D.
5. Use of surfactants and solvents to improve
availability of contaminants
5.1 Advances on hysteresis characterization
Hysteresis coefficient is used for determining the adsorption-desorption behavior of pollutants on sediments and
soils. In soil and sediment pollution, the adsorption and
desorption play a key role on pollutants availability and
transport. For several pollutants and solid matrices, the
desorption pathway is different from that of adsorption.
This phenomenon is known as hysteresis. Poggi-Varaldo
et al. [56] have defined a hysteresis coefficient CH as the
ratio of the slope (derivative) of the adsorption curve and
the slope of the desorption curve in a given point (Cj, qj)
of interest. When hysteresis is not important, CH ≅ 1, i.e.,
the adsorption is reversible, when hysteresis is important,
then CH >1, i.e., the adsorption is irreversible. Among several uses of the CH, it can be cited the following: quantification of the effect of aging on the availability of a
pollutant adsorbed onto sediments or soils; quantitative
comparison of the degree or retention of a pollutant in
several types of solid matrices; quantification of the effect
of adding surfactants and solvents to a given system pollutant-solid matrix; quantitative comparison of the degree
or retention of several pollutants in a given sediment or
Table 4: Anaerobic slurry bioreactors.
Matrix
Contaminant
Soil
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
Soil
Soil
sediment
Soil
Soil
Soil
Soil
Soil
Soil
Soil
Remarks
Removal Ref.
With anaerobic activated sludge as the microbial inocula and
sludge cakes as the primary substrates
Chlorpyrifos
Sequencing batch mode (anoxic-aerobic-anoxic)
Hexahydro-1,2,3-trinitro-1,3,5-triazine (RDX) With supplementation of municipal anaerobic sludge as an
exogenous source of microorganisms
PAH Acenaphthene
Addition sulfate as an electron acceptor enhanced PAH
degradation
αβδ and γ-hexachlorocyclohexane
Bioaugmented with anaerobic sludge
2-sec-butil-2,6-dinitrofenol (DINOSEB)
Pilot scale
2,4-dichlorophenoxy-acetic acid
Sulfate reduction conditions with addition of sucrose
Pentachlorophenol (PCP)
Bioaugmented with cells of Desulfitobacterium frappieri strain PCP-1
Trinitrotoluene (TNT)
Augmentation with anaerobic biomass from a food industry
wastewater treatment plant
Diesel
a)mixed electron acceptor (SO4=, NO3-, CO2)
b)sulfate-reducing condition
Tetrachloroethylene
Bioaugmented with cells of Desulfitobacterium sp. strain Y-51 and
addition of zero.valent iron (Fe0)
86%
[112]
91%
60%
[113]
[114]
79%
[115]
100%
51%
48%
100%
100%
[68]
[55]
[30,31]
[87]
[116]
a)80%
b)55%
100%
[29]
[117]
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soil; and estimation of the retardation factor of transport
of contaminant in porous, adsorbent media [31,56].
The availability enhancement factor (AEF) is used for
determining the effectiveness of desorption treatments of
pollutants from soils and sediments. The AEF was defined
as the ratio of the slope of the desorption curve of a given
pollutant with a surfactant or solvent treatment to the
slope of the corresponding curve of the reference treatment (usually desorption with distilled water). This factor
has proved to be useful for the quantification of the effect
of a given soil treatment (with surfactant, biosurfactant,
solvents) on the possible improvement of the pollutant
desorption and availability for further biodegradation or
physicochemical removal [57].
5.2 Surfactants
Surfactants may facilitate hydrophobic pollutant desorption from a solid matrix, and its dispersion in the aqueous
phase. They may also help in stabilizing the slurry soilaqueous medium in the SB. Surfactants are classified as
anionic (SDS, LASA and SDOSS), cationic (benzyl trimethyl-ammonium bromide) and non ionic (Triton X-100;
Brij 35; Tergitol NP-10, Tween-80, etc.) from the point of
view of predominant electric charge after contact with
water [58,59]. They can also been classified as synthetic
and biological surfactants, according to its origin or production. The first ones are mainly manufactured by petrochemical plants, whereas the second ones are produced by
living organisms. Examples in the second category are
rhamnolipids, glycolipids or lipoproteins, among others
[60-65].
Main factors that determine surfactant success and capability are its chemical nature and concentration as a multiple of critical micellar capacity (CMC). For synthetic
surfactants, it has been observed that although they may
be very effective for increasing the detachment of pollutants from soils, they usually become toxic to microbes at
the high concentrations required for their action; very
often pollutant degradation is adversely affected
[35,36,64,66,67]. Results on surfactant capability and performance in the literature has been obscured by experimental designs that have not taken into account the
surfactant CMC [58]. In this way, several works report no
effect or negative effect of surfactants on pollutant mobilization and detachment from the solid matrix. Closer
examination of a large amount of these experiments have
revealed that surfactants were used at concentrations
lower than their corresponding CMCs. Now it is generally
recognized that surfactants should be added at a concentration higher than its CMC in order to effectively disperse
and pseudo-solubilize the hydrophobic pollutant in
water.
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Regarding synthetic surfactants, the order of preference
generally accepted is: non ionic > anionic > cationic. Cationic surfactants are usually toxic to microorganisms and
also they can adsorb to negatively-charged sites of the clay
and organic matter of the soil, reducing the actual concentration value. So, it is recommendable not only to carry
out in vitro studies for determining surfactant capability
[57] but inhibition studies of the candidate surfactants on
the active microflora of the SB as well [68].
Biodegradable surfactants and biosurfactants may be the
choice for increasing pollutant availability while minimizing inhibitorial effects on SB microflora. Yet, some
biodegradable surfactants can compete with the pollutant
as carbon source for the microorganisms provoking a preferential degradation of the surfactant and a lower removal
of the contaminant [35,36,67,69]. Microbial surfactants
have proved to be able to combine excellent hydrophobic
pollutant-mobilizing properties with other special features, rarely displayed by commercial chemical surfactants, such as complete biodegradability and nontoxicity [62,65]. Unfortunately, little is very often known
about their chemical-physical properties, and they are still
not available on the large scale at low price, and this currently precludes their application in bioremediation. It
has been recently shown that some commercial mixtures
of biogenic compounds, such as cyclodextrins, phytogenic
surfactants of water-soluble fraction of humic substances,
can markedly intensify the bioremediation of PCB-, PAHand/or hydrocarbon-contaminated soils in SBs (Tables 1
and 2) by displaying the same advantages showed by
microbial surfactants. In particular, Fava and co-workers
[45,46] demonstrated that two cyclodextrins (CDs), i.e.,
hydroxypropyl-β-CD and γ-CD, and the phytogenic surfactant Quillaya Saponin were able to enhance from 15 to
30% the aerobic bioremediation of an actual site, aged
PCB-contaminated soil (with 350 mg/kg of total PCBs)
when applied at 10 g/l under slurry-phase conditions. The
agents did not exhibit the toxicity and the recalcitrance
displayed by the chemical surfactants Triton X-100 tested
at the same concentration in identical parallel laboratoryscale SBs. Then, the same group [70-72], investigated the
opportunity to use less costly industrial CD products, and
in particular a mixture of RAndomly MEthylated-β-cyclodextrins (RAMEB), to intensify the aerobic bioremediation of 5 other different PCB-contaminated soils under
several treatment conditions including laboratory-scale
SBs. A faster and more complete removal of PCBs was generally observed in the amended bioreactors for the ability
of RAMEB to improve both PCB occurrence in the soil
water-phase and the growth and the occurrence of soil
specialized bacteria in the reactors. Larger PCBs biodegradation and dechlorination, along with larger toxicity
depletion, were generally observed in SBs than in the
solid-phase or saturated soil loop reactors, especially in
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the presence of RAMEB. This may be ascribed to the
higher degree of homogeneity and higher mass-transfer
rates typical of slurry-phase conditions [20,21] which, in
turn, probably enhance the intimate contact among the
specialized microorganisms, the mobilizing materials and
the soil-sorbed PAHs. Then, the beneficial effects of
RAMEB have been also demonstrated in the aerobic bioremediation of PAH- and transformer oil-contaminated
soils under laboratory slurry and solid-phase conditions
as well as in the field, through pilot scale experiments conducted both under ex-situ (biopile) and in situ conditions
[73]. Recently, the potential enhancing effects of other
two commercial biogenic pollutant-solubilizing agents,
namely a technical mixtures of Soya Lecithins and an
extract of water soluble humic substances of North
Dakota Lignite, on the aerobic slurry-phase bioremediation of a model soil spiked with PCBs [74,75] and of an
aged PAH-contaminated soil [11] have been demonstrated. In the latter case, a soil historically contaminated
by about 13 g/kg of a large variety of PAHs was amended
with soya lecithin or humic substances at 1.5% w/w and
treated in aerobic solid-phase and SBs for 150 days. The
overall removal of PAHs in the presence of the agents was
faster and more extensive and accompanied by a larger
soil detoxification, especially under slurry-phase conditions. The agents could be metabolised by soil aerobic
microorganisms and enhanced the occurrence of both soil
PAHs and indigenous aerobic PAHs-degrading bacteria in
the reactor water-phase. Thus, the agents were biodegradable and efficiently enhance PAH biodegradation by
improving the availability of both PAHs and specialized
microorganisms in the soil reactors.
compounds and this makes their incorporation into soil
more socially acceptable than that of the chemical surfactants currently proposed to intensify the bioremediation of aged PCB- and hydrocarbon-contaminated soils.
All biogenic pollutant-mobilizing agents mentioned
above were found to be promising bioremediation
enhancing additives, as capable of combining marked
enhancing effects on the process with a complete biodegradability, non-toxicity and therefore a complete environmental compatibility. In addition, they are biogenic
For successful application of either 3P or 2P-SB technology, appropriate selection of the solvent is of paramount
importance: solvent should be immiscible in water, non
toxic to microorganisms, non biodegradable, stable and
non flammable, non volatile, and with high affinity for
hydrophobic pollutants [78]. According to Villemur et al.
5.3 Solvents
Solvents can also be used for increasing the availability
and bioavailability of low solubility-, hydrophobic pollutants in soil remediation. In particular, immiscible (in
water) and non degradable solvents with affinity for
hydrophobic contaminants can help in attracting the molecules of contaminants adsorbed onto soil, transfer the
contaminant into the solvent phase, and afterwards to
facilitate the exchange of contaminant between the solvent to the aqueous phase [6,76] where microorganisms
can finally degrade the pollutant. In SB practice, there is
little or no experience on using solvents for this purpose.
If it were done, the SB would be a tri-phasic bioreactor
(3P-SB) with one particular solid phase (soil and microorganisms) and two liquid phases (droplets of solvent dispersed in the water phase).
Available information mainly comes from the application
of two-phase 2P (liquid, solvent-water) bioreactors (Table
5). Knowledge accumulated from the 2P (liquid) bioreactor practice indicates that best solvents used for enhancing
biodegradation of PAH are paraffin oil, silicone oil, n-hexadecane, corn oil, cis-jasmone, r-limonene, 2-undecanone, l-decene and n-dodecane [6,64,77]. The 2P
bioreactor concept has been shown to be very effective for
biodegradation of high concentrations of toxic organic
compounds by bacteria, Table 5.
Table 5: Biphasic and triphasic bioreactors for treatment of hydrophobic organic pollutants (adapted from [78]).
Pollutant
Microorganisms
Solvent
Styrene
Phenantrhene
Naphthalene
Various PAHs
2,4,6-trichlorophenol
Dioxins
Pentaclorophenol
Phenol
Phenanthrene
Benzene
Benzene
Benzene
Mixed culture
Pseudomonas aeruginosa
Corynebacterium sp.
Mixed culture
Pseudomonas sp.
Mixed culture
Arthrobacter sp.
Pseudomonas putida
Pseudomonas sp.
Alcaligenes xylosoxidans
Klebsiella sp.
Alcaligenes xylosoxidans
silicone oil
2,2,4,4,6,8,8-heptametilnonane
Decane, dodecane, hexadecane
silicone oil
silicone oil
Decane
Diethyl sebacate
2-undecanone
silicone oil
Hexadecane
1-octadecane
Hexadecane
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[38] and Collins & Daugulis [79] 2P bioreactors might
provide an efficient way to bioremediate contaminated
soils. For example, in a large scale process, a 2P-SB (soilsolvent) could be used to extract pollutants from a contaminated soil by transferring to the water-immiscible,
non degradable liquid phase. The solvent would then be
recovered and the treated soil returned to the site. This
could be repeated a number of times with the same solvent. Once the water-immiscible liquid has reached a certain concentration of contaminant, the extraction vessel
could then be used as a 2P-bioreactor (liquid, solvent and
water) to degrade the extracted contaminants.
6. Bioaugmentation and advances on
characterization and monitoring of microbial
communities of slurry bioreactors
Very often SB operation relies on the use of native microflora already existing in the polluted soil. However, whenever native microflora is scarce or weak or with no
apparent capability of degrading the target compounds, it
is advisable to inoculate SB with enriched/acclimated consortia or strains, more commonly consortia. This is called
bioaugmentation. Thus, inoculation of 'specialized' biomass may allow for an increased biodegradation of target
pollutants as well as a more effective detoxification of the
solid matrix [47,80,81]. Another common result of bioaugmentation is the dramatic reduction of remediation
times [5].
Barbeu et al. [82] have recommended a protocol for producing aerobic acclimated consortia based on activated
sludge process. An outline of their procedure follows: start
a complete-mix activated sludge reactor with 10% (w/v)
of soil and a mineral medium. The concentration of the
target pollutant(s) in the feed of bioreactor is cautiously
increased step-wise until the desired concentration in the
feed is reached. After 30 days of operation, provided that
reactor monitoring indicates the uptake of the contaminant, an acclimated consortium is developed with the
capability of using the target pollutant(s) as energy and
carbon sources. Acclimated inocula can also been
obtained from polluted sites such as sediments, soils and
wastewaters [6,9,64]
To a lower extent and mainly in laboratory and pilot scale
trials, specific strains have been used for bioaugmentation. A three-membered co-culture of strains of Pseudomonas sp and Alcaligenes sp. have been applied to
aerobic degradation of PCBs [47]; Sphingomonas aromaticivorans to degradation of PAHs [6]; Flavobacterium sp. for
degradation of naphtalene [9]Burkholderia cepacia JS872,
B. cepacia and Hydrogenophaga palleronii for degradation of
2,4-DNT and 2,6-DNT [5]. However, several failures have
been recorded in this field; they have been mostly
ascribed to the inability of inoculated specialized pure or
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mixed cultures to compete with autochthonous microflora and to face the toxicity and the scarcity of nutrients
occurring in the contaminated biotope [83]. Thus, the
opportunity to bioaugment contaminated soils with complex microbial systems consisting of a large variety of
robust microorganisms and essential nutrients has been
recently explored [33,84]. In particular, Di Toro et al. [85]
studied the effects of Enzyveba, i.e. a partially characterized consortium of microorganisms developed from the
stabilization of high quality organic wastes, on the aerobic
bioremediation of an actual-site aged PCB-contaminated
soil (overall PCBs: 920 mg/kg) under laboratory-scale
slurry and solid-phase conditions. Markedly enhanced
PCB-biodegradation rates and extents (from 50 to 100%)
and soil detoxification as well as higher availability and
persistence of aerobic PCB- and chlorobenzoic aciddegrading cultivable bacteria were observed in the Enzyveba-amended SBs. These findings suggested that Enzyveba enhanced the biotreatability of the selected soil by
providing exogenous bacteria and fungi able to remove
inhibitory or toxic intermediates of PCB biodegradation
and/or exogenous nutrients able to sustain microorganisms in charge for PCB mineralization.
Also, dehalorespiring strains that use organo-chlorinated
compounds as electron acceptors have used in bioaugmentation such as Desulfitobacterium frappieri PCP-1 and
Desulfitobacterium dehalogenans introduced into non-sterile soil or soil slurry microcosms [86,87].
Monitoring of microbial communities and specific strains
in soil SBs is becoming an important tool for improving
our understanding of microbial population dynamics
[88], the fate of inoculated cultures as well as biodegradation mechanisms, and the influence of process operating
conditions and other variables [87].
Monitoring of microbial community is traditionally
accomplished by microbial counting by total or selective
plating, most probable number counting, or bacterial
staining followed by microscopic analysis, or by measuring microbial activity through respirometric or radiorespirometric analyses [89]. Due to experimental
limitations, such as the lack of suitable electron donors or
acceptors or of nutritional factors in the growth media or
the unsuitable growth conditions as regards temperature,
pressure or redox conditions, most of these methods are
inadequate to give a reliable picture of the microbial community. However the most important factor affecting the
reliability of methods based on cultivation of microorganisms is the poor culturability of most of microorganisms
in complex environments such as those created in the laboratories [90]. When the active microbial population is
poorly cultivable the effectiveness of a remediation process cannot be efficiently predicted or followed during a
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treatment by using traditional microbiological methods.
Modern molecular biology tools offer in these cases several advantages respect to traditional microbiological
methods, since they can couple qualitative and quantitative information on the microbial community including
the uncultivable fraction. The polymerase chain reaction
(PCR) and competitive PCR (cPCR) have been used with
success to monitor Desulfitobacterium frappieri PCP-1 and
Desulfitobacterium dehalogenans introduced into non-sterile soil or soil slurry microcosms [86,87]. A previous
report showed that strain PCP-1 can dechlorinate PCP in
anaerobic soil slurry microcosms and can be monitored
by PCR when introduced in this system [91]. The fatty
acid methyl ester (FAME) analysis approach was successfully employed by Cassidy & Hudak [92] to selectively
monitor the main bacterial and yeast strains responsible
for biosurfactants production in a 8 L slurry-sequencing
batch reactor developed to treat a soil contaminated with
21.8 g/kg diesel fuel. The results of the study demonstrated that SB operation can be manipulated on the basis
of microbial monitoring information to control and
improve the overall reactor performance [92]. No other
reports on microbial molecular/biochemical monitoring
of SBs were found in the literature. However, such
approaches might allow predicting, following and
improving the effectiveness of SB bioremediation processes, and therefore they should be included in the integrated strategies for SB monitoring and assessment.
7. Developments on ecotoxicity assays aimed at
evaluating bioremediation efficiency of the
process
Several types of ecotoxicity bioassays are known, some of
them have already been standardised, some others are
under development [93,94]. They are carried out in polluted and treated soil to further assess treatment performance and end point. Information of these tests shed light
on "true" soil detoxification achieved by treatment, in
opposition of "apparent" decontamination assessed by
mere chemical or instrumental analysis. In effect, pollutant removal sometimes may be accompanied by transformation of the mother contaminant into toxic
intermediates that could persist in soil. So, contaminant
removal not always is equivalent to decontamination or
detoxification [95]. Ecotoxicity tests also account for the
interactions among pollutants, which might interact
through a synergic or antagonistic manner. Again, this
type of interaction cannot be observed by mere instrumental analysis [96]. Thus, ecotoxicity assays appear as a
significant complementary tool to assess the extent and
success of bioremediation [97].
Some ecotoxicity tests are carried out using the aqueous
extracts of soils: they are useful when pollutants are water
soluble. The OECD [98-102] has proposed to apply a vari-
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ety of tests organisms belonging to different levels of the
trophic chain: microorganisms (Pseudomonas putida,
Photobacterium phosphoreum), primary consumers (micro
crustaceans Daphnias); secondary consumers (fish); primary producers (algae).
Yet, some other tests are performed using whole soil.
These methods are also quoted as direct contact tests, as
they rely on the growth or germination of well defined,
artificially cultivated test-organisms directly into the contaminated soils. Test organisms include plants (Brassica
rapa, Avena sativa, Lepidium sativum, etc), seeds for assessing germination, and mortalitiy of earth worms (Eisenia
fetida, Eisenia andrei) [103]. These tests are particularly
indicated for those soils contaminated by hydrophobic
pollutants such as PAHs and PCBs, which typically are
sorbed onto the soil. Other ecotoxicity assays rely on the
evaluation of biological abundance or diversity of organisms autochthonous of the contaminated soils (nematodes, microorganisms) [97,100,104] or in the measure of
some specific activities of the soil, like respiration,
enzyme activities, etc. [105].
A combination of the Lepidium sativum roots and shoots
elongation inhibition test and of Folsomia candida mortality test has been successfully applied by Fava and co-workers in the assessment of the efficacy of different slurryphase bioremediation strategies developed for actual-site
soils contaminated by PCBs [14,47,71,72,74,75,85] and
PAHs [11]. The same tests along with other contact tests
based on the use of selected bacteria have been applied in
the monitoring of the toxicity of hydrocarbons-contaminated soils subjected to bioremediation under slurry but
also solid-phase pilot-scale conditions [73].
Conclusion
SB is an ad situ and ex situ technology that can be used for
bioremediation of problematic sites (when the less expensive natural attenuation or stimulated in situ bioremediation are not feasible), such as those characterized by soils
with high contents of clay and organic matter, contaminated with pollutants that are recalcitrant, toxic, and display hysteretic behavior, or when bioremediation should
be accomplished in relatively short times under strong
pressure and monitoring of environmental agencies and
regulators. SB technology allows for the convenient
manipulation and control of several environmental
parameters that could lead to enhanced and faster treatment of polluted soils: nutrient N, P and organic carbon
source (biostimulation), inocula (bioaugmentation),
increased availability of pollutants by use of surfactants or
solvents, or inducing biosurfactant production inside the
SB, etc. An interesting emerging area is the use of SB with
simultaneous electron acceptors, which has demonstrated
its usefulness for the bioremediation of soils polluted
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with hydrocarbons. Characterization studies of microbial
communities of SB are still in the early stages, in spite of
their significance for improving reactor operation and
design optimization; so far SB are still modeled as "black
boxes".
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3.
4.
5.
Inter alia, we have identified the following avenues of
research for SB in the future: (i) application of SB with
sequential and simultaneous electron acceptors to soils
polluted with contaminants other than hydrocarbons
(i.e., pesticides, explosives, etc.), (ii) evaluation of the
technical feasibility of triphasic SB that use innocuous solvents to help desorbing pollutants strongly attached to
soils, and in turn, to enhance their biodegradation, (iii)
gaining deeper insight of microbial communities present
in SB with the intensified application of molecular biology tools such as PCR-DGGE, PCR-TGGE, ARDRA, etc.,
(iv) development of more representative ecotoxicological
assays, more complex however more informative than the
mere uni-species bioassays or battery of uni-species tests
(for instance, microcosm and mesocosms bioassays) to
better assess the effectiveness of a given bioremediation
process.
Abbreviations
A: Aerobic; AEF: Availability enhancement factor; CMC:
Critical micellar capacity; CDs: Cyclodextrins; 2,4-D: 2,4dichlorophenoxyacetic acid; 2,4-DNT: 2,4-dinitrotoluene;
2,6-DNT: 2,6-dinitrotoluene; DEP: di-ethyl phthalate;
EPTC: S-ethyl dipropylthiocarbamate; ETP: Effluent treatment plant; HCH: Hexachlorocyclohexane; CH: Coefficient of hysteresis; LO: Lubricating oil; PCR: Polymerase
chain reaction; PAHs: Polynuclear aromatic hydrocarbons; PCB: Polychlrorinated byphenyls; TPH:Total petroleum hydrocarbons; 2,4,6-TNB: 2,4,6-trinitrobenzene;
2,4,6-TNB 2,4,6-trinitrotoluene; SB: Slurry bioreactor;
TLP: Three-phase bioreactor; TPH: Total petroleum hydrocarbons
Authors' contributions
All authors participated equally in the conception and
writing of this review. All authors read and approved the
final manuscript.
Acknowledgements
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The authors wish to thank CONACYT for a graduate scholarship to IVRG, and CINVESTAV. The insightful comments and suggestions of the Editor
and the anonymous Referees of Microbial Cell Factories are gratefully
acknowledged.
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