R

Remediation
di ti off humic
h i soilsil contaminated
t i t d with
ith
benzene usingg bioremediation
M. Rosas1, V. Domingues
g 1, P. Marco3, T. Oliva‐Tele
es1, A.Fiuza2, J. T. Albergaria
g 1 and C. M. Delerue‐Matos1

1 REQUIMTE, Instituto Superior de Engenharia do Porto,

REQUIMTE Porto Rua D
Dr António Bernardino de Almeida 431
Dr. 431, 4200‐072 Porto,
Porto Portugal.
Portugal
2 CIGAR,
CIGAR Faculdade
F ld d de d Engenharia
E h i da
d Universidade
U i id d ddo Porto,
P t RuaR Dr.
D Roberto
R b t Frias,
F i 4200‐465
4200 465 Porto,
P t Portugal.
P t l
3IBMC, Universidade do Porto, Portug
gal and CICS
CICS‐ISCSN,
ISCSN, Gandra PRD, Portugal.

INTRODUCTION
The release
Th l off contaminants
t i t into
i t the
th soilil matrix
t i produces
d a negative
ti impact
i t in
i holding
h ldi capacity it (field
(fi ld capacity)
it ) off the
th soilil or about
b t 12 percentt to t 30 percentt The workk presented
Th t d represents
t a partt off a wider
id project
j t whose
h main
i goall is
i
soil quality, creating, in some cases, situations of risk to public health. To invert by weight. In general, the soil should be b moist but not wet or dripping wet. the development of an artificial neural network able to predict the time
this situation several remediation technologies g can be used,, such as Excessive soil moisture,, however,, restrricts the movement of air through g the needed to remediate a soil contaminated with p popular
p contaminants such as
bioremediation, in situ oxidation, soil vapour extraction or even the subsurface thereby reducing the availab bility of oxygen which is also necessary benzene, toluene, ethylbenzene, xylene, trichloroetylene or perchloroethylene
combination of two or more technologies.
technologies Bioremediation,
Bioremediation that is one of the for aerobic bacterial metabolic proceesses. esses Microorganisms need inorganic combining soil vapor extraction and bioremediation.
bioremediation The objectives of this part
most usedsed remediation technologies
te hnolo ies in the United States [1], [1] uses
ses the n trients such
nutrients s h as nitrogen
nitro en and phosphorus or s to ensure
ens re cell
ell growth
ro th and sustain
s stain of the work
ork are to evaluate
e al ate the capability
apabilit of bioremediation to achieve
a hie e legal
le al
d
degradation
d capacity off indigenous
d or inoculated
l d microorganisms to b
bioremediation
d processes. However, exccessive amounts off those h nutrients can clean‐up
l goals
l in humic
h soils
l contaminated d withh benzene
b previously
l
biodegrade
g organic
g constituents adsorbed to soils. It is a technology gy that repress
p microbial metabolism. The typ yppical carbon:nitrogen:phosphorus
p g p p ratio remediated byy soil vapor
p extraction,, calculating:
g
generally needs long periods of treatment to reach the desired clean clean‐up
up goals. necessary for biodegradation falls in the range of 100:10:1 to 100:1:0.5, i) process efficiency and ,
The process requires some nutrients and moisture in the soil; in some cases depending upon the specific constituen nts and microorganisms involved in the ii) remediation time; and iii) to select microorganisms that efficiently degrade
these requirements are present in the soil matrix but in other cases it is biodegradation process [2]. [2] benzene
benzene.
necessary to introduce
i d them
h i the
in h soil.
il Iff allll these
h needs
d are satisfied,
i fi d biorem
bi mediation
di i is i efficient
ffi i to degrade
d d toxici To achieve
hi these
h objectives
bj i severall experiments
i were performed
f d in
i a humic
h i
The ideal range g for soil moisture is between 40 and 85 p percent of the water‐ organic
g contaminants such as benzene. soil with an organic
g matter content (OMC) of 14%.

MATERIALS AND METHODS

SOIL PREPARATION INOCULUM PREPARATION BIOREMEDIATION TESTS
FLASK COLUMN PROCESS MONITORING
Sandy Soil Washing Humic Soil Bacterial growth
Minimal medium ((MinE))
0 2% carbon source
0.2%
D i att 110ºC
Drying D i att 50ºC
Drying d i 6 days
during d att 23ºC

Sieving Sieving
Centrifugation
REJECT
Above 2mm Ab
Above 2
2mm
Below 2mm Below 2mm ‐ 30 g off soil,
il ‐ 2 .0 kgg of soil,,
15 mL inoculum
i l ‐ 15 mLL off tthe
th singular
i l bacterial
b t i l cultures
lt All the bioremediation tests were
‐ 800 mL of water,,
(Pseudomonas fluorescens (PfST), monitored d b
by analysing
l the
h
Soil with 14% OMC ‐ 15 mL off the
h singular
l bacterial
b l concentration of benzene in the gas
Pseudomo onas putida KT2440 (PpKT),
(PpKT)
culture of ST,
ST phase
h th
through
h gas chromatograpy
h t
Pseudomo onas stutzeri ((OX1),
) Labrysy
RESULTS portucalensis (F11)),
(F11)) ‐ contamination of 70, 90 and 120 usingg a flame ionisation detector.
mg kg‐11 of benzene
here at 40 mg L‐1 of benzene
‐ atmosph

MICROORGANISM SELECTION COLUMN TESTS

The first step was to select the most efficient The legislation impose that a soil is considered contaminated with Three soils previously treated with SVE and with different levels
microorganism
g capable
p do degrade
g the benzene. benzene if the level of contamination is higher
g than 10 mgg kgg‐11. A of contamination ((70,, 90 and 120 mgg kgg‐11) were tested. The
Figure 3 shows how the four microorganisms column was prepared with that level of conttamination and the bioremediation monitoring is presented in Figure 5.
5
performed
f d in
i an atmosphere
t h off 40 mg L‐11 off concentration
t ti off the
th contaminant
t i t in
i the
th gas ph
h
hase was monitored
it d
benzene. through time until equilibrium was reached.
20 6,0
Sterile soil
0,75
Non sterile soil 5,0
15
PfST 120 mg kg
kg-11
mg L-1)

0,60 4,0
mg L-1)

PpKT
p
90 mg kg-1
Cggas ((mgg L-1)

OX1
Cgaas (m

30
3,0
Cgass (m

10 0,45
F11 70 mg kg-1
kg 1
20
2,0
0,30
5
1,0
,
0,15

0 0,0
0 2 4 6 8 10 0 50 100 150 200 250 300 350 400
0,00
0 50 100 150 200 250
Time (h)
Time (d)
Time (h)
Figure 3 ‐ Flask tests to select the most efficient Figure 5 ‐ Column
Fi C l tests performed
f d in
i soils
il with
i h three
h
microorganism
microorganism. levels of contamination.
Fi
Figure 4 ‐ Column
C l test in
i soilil with k ‐1
i h 10 mg kg‐1 off benzene.
b

The microorganisms chosen for the follow

column
l t t was Pseudomonas
tests P d fl
fluorescens d
due Th SVEs
The SVE will
ill be
b stopped
t d when
h Cgas reach
h th 0 40 mgLL‐11.
h 0.40
he To reach the legal limit,
limit the bioremediation of these soils
the higher rate to degrade benzene . required
i d 92,
92 139 andd 354 hours
h respectively
ti l for
f the
th 70
70, 90 and
d
120 mg kg‐1 soils.
CONCLUSIONS

The results obtained allowed concluding that:

a)) bioremediation
bi di ti isi an efficient
ffi i t technology
t h l t remediate
to di t soils
il similar
i il to t those
th experimented
i t d contam
t minated
i t d with
ith benzene,
b
b)) all the experimented
p microorganisms,
g , combined with the soil,, showed ggood degradative
g capacity;
p y;; the Pseudomonas ffluorescens was chosen,,
c) the soil,
soil as it was prepared,
prepared showed also good capacity to degrade benzene,
benzene
d) the
h bioremediation
b d times for
f theh soils
l previously
l remediated
d d with h SVE where
h 92, 139 and
d 354 hou
h urs for
f soils
l contaminated
d with
h 70, 90 and k ‐11 respectively.
d 120 mg kg l

ACKNOWLEDGEMENTS REFERENCES

The
h authors
h are grateful
f l to Fundação para a Ciência
ê e [1] United STATES Environm mental Protection Agency,
Agency Treatment technologies for site cleanup: Annual Status Report (Twelfth
Tecnologia (PTDC/ECM/68056/2006) for the material Edi i ) 2007,.
Edition), 2007 Retrieved
R i d Oc October
b 5,
5 2007 from
f h // l i
http://clu‐in.org/asr/.
/ /
supportt for
f this
thi work.
k [2] USEPA, United STATES Environmental Protection Agency, How To Evaluate Alternative Cleanup Technologies For Underground
Storage Tank Sites: A Guide For Corrective Action Plan Reviewers
Reviewers, Chapter V
V, (1994)
(1994).
http://www.epa.gov/swerus
p // p g / st1/pubs/tum
/p / _ch5.pdf.
p ((accessed 10/03/2009).
/ / )