Tema 1C – Remediation Concepts & Technologies

PhD Work: Yes

EXTENSIVE METHODOLOGY FOR PRELIMINARY BIOVENTING TESTS – APPLICATION TO A

RESIDUAL GRANITIC SOIL CONTAMINATED WITH XYLENE

Manuela Carvalho. (1),(2); M.Cristina Vila (2); José Soeiro de Carvalho (2); Valentina Domingues (1);
Cristina Delerue-Matos (1); Teresa Oliva-Teles (1);António Fiúza (2)

(1) REQUIMTE, Instituto Superior de Engenharia do Porto, Rua Dr. António Bernardino
de Almeida 431, 4200-072 Porto, Portugal, +351228340500, mmc@isep.ipp.pt
(2) CIGAR, Faculdade de Engenharia da Universidade do Porto, Rua Dr. Roberto Frias,
4200-465 Porto, Portugal, +351225081989, afiuza@fe.up.pt

Keywords: Bioventing, xylene biodegradation, kinetics tests, residual granitic soils

Abstract

Bioventing is an in-situ remediation technology that uses indigenous microorganisms to biodegrade

organic constituents adsorbed to soils in the unsaturated zone, being the required oxygen supplied by
circulation of air induced by pressure gradients. It promotes biodegradation of contaminants
minimizing volatilization. Compared to Soil Vapour Extraction (SVE), the amount of volatilized
contaminant is negligible; bioventing has the advantage of being less harmful to the atmosphere
avoiding the need for an exhaustion gas phase treatment.

A new laboratory experimental methodology for the preliminary assessment of a bioventing system is
proposed and thoroughly explained. It involves a detailed check-up of the relevant specific properties
of the soil and contaminants, the characterization of the autochthonous heterotrophic microorganisms
and the operating conditions to be implemented.

The effectiveness of biodegradation was studied in a residual granitic soil. This soil, predominantly
sandy-silt, is common in the subsurface of the Portuguese territory.

The microorganisms consortium was collected from the protection area (retention basin) of crude and
diesel storage tanks in a refinery. Xylene was the pollutant elected to describe the methodology
procedures.

Extensive kinetics studies in flasks allowed determine the most appropriate range for the growth of the
degrading microorganisms for different concentrations of the contaminant. The higher bacterial growth
was obtained with a concentration of xylene between 0.16 and 0. 24 µL per mL of inoculum. It was
also verified that the incubation temperature was not a relevant parameter. The developed consortium
showed high efficiency (≥ 98%) in degrading xylene.

Gas Chromatography (GC) with flame ionization detector (FID) was used to control contaminant
concentration in the gas phase and the biomass was measured by counting colony forming units
(CFU).
1. Introduction

Bioventing is an in-situ remediation technology combining SVE with bioremediation principles. It is

used on semi-volatile organic compounds. It consists in a forced air circulation through a contaminated
soil by vacuum-pumping in an extraction well. At the same time it promotes the contaminants
biodegradation by autochthonous microorganisms in the infiltration zone of the soil [1, 2, 3].

Bacteriological activity is usually stimulated by air or oxygen flux through the infiltration zone, and,
when needed, by added nutrients. The two outstanding parameters for a suitable bioventing of a
contaminated area are: i) the soil permeability which determines the withdrawal of contaminant from
the gaseous phase and also the rate of oxygen delivery to aerobic heterotrophic bacteria
(biodegrading agents) and ii) the contaminant biodegradability determined by the metabolism of the
microorganisms [1].

The aim of this work was to study bioventing viability to remove hydrocarbon from contaminated
residual granitic soils, using a consortium of aerobic bacteria obtained from contaminated soils. Mixed
microbial communities have more powerful biodegradative potential than a single strain [4]. The
effectiveness of bioventing was tested in a residual granitic soil. This soil, predominantly sandy-silt, is
common in the subsurface of the Portuguese territory, particularly in the north.

Xylene was selected to initiate the systematic study of diesel remediation, as it widely spread in soils,
sediments and groundwater near diesel/gasoline service stations and refineries.

In this work the methodology established for preliminary bioventing tests was:

• The residual granitic soil (Srγ) was sampled and characterized by the main physical, chemical
and geotechnical properties;

• A soil sample contaminated with hydrocarbons (BSoil) was collected from the protection area
(retention basin) of crude and diesel storage tanks in a refinery. Extraction of the bacteria
consortium from this soil and bacterial consortium enrichment for biodegradation of xylene
was performed;

• Kinetics studies of xylene biodegradation were previously done in liquid medium with various
contamination levels (substrate) and constant temperature to select the most adequate
working concentration range. Macro and microscopic characterization of the microorganisms
in the bacterial consortium, at various xylene concentrations was made;

• Kinetics studies of xylene biodegradation in a residual granitic soil, using the previously
selected concentrations of contaminant and different temperatures;

• Test of the capability of the bacterial consortium selected to biodegrade the xylene in the Sry
using continuous lab-scale equipment.

2. Methodology

2.1. Selection, sampling and soil characterization

“BSoil”
3
BSoil sample (approx. 20 dm ) was collected and stored in a metal container at room temperature and
protected from light. Visually, it was a soil with extended grain size distribution, with a fine clay fraction
and a high amount of organic matter. The determination of total petroleum hydrocarbons (TPH) was
performed using the colorimetric method (Remediaid test kits from Chemetrics) with a result value of
14 686 mg of TPH per kilogram of soil.

Residual Granitic Soil (Srγ)

The soil sampled and used in this study (Figure 1) is a soil resulting from weathering of the “Porto
Granite” and was collected on a recent slope excavation, at 2-3 m depth. Visually it was a residual
granitic soil of medium particle size, oxidized and kaolinized.

Figure 1 – A sample of the granitic soil studied.

The geotechnical characterization (particle size distribution, density, bulk density, porosity, maximum
density, optimal water content), pH, conductivity at 25ºC and organic matter was based on standard
methods [5, 6, 7, 8, 9].

2.2. Selection of the bacterial consortium

The cultures were done in sterilized Erlenmeyer flasks (500 mL) closed by Mininert valves and
incubated at 28°C ( WTW – TS1006-i), with shaking (150 rpm; Heidolph – Unimax 1010).

Enrichment cultures were obtained by adding “BSoil” (10 g) to the mineral liquid medium (100 mL).
Several transfers were performed from the enrichment cultures. The first transfer was 5% (V/V)
inoculum and each subsequent one was 50% (V/V). Xylene was added to each transfer as a carbon
source. Each enrichment was tested with different amounts of xylene in order to define the tolerance
of bacteria to contaminants. Seven different quantities were studied, ranging from 0.04 to 0.4 µL of
xylene per mL of inoculum. The period between transfers was variable, depending on the kinetics of
contaminant degradation in each flask.

The composition of the mineral liquid medium was (NH4)2SO4 (3.8 mmol/L), KNO3 (1.02 mmol/L) and
NaNO3 (8.21 mmol/L) as nitrogen sources. Furthermore, the medium contained Na2HPO4 (6.0
mmol/L), KH2PO4 (4.0 mmol/L), CaCl2.H2O (0.47 mmol/L), NaCl (0.14 mmol/L), MgSO4.7H2O (0.41
mmol/L), nitrilotriacetate (0.52 mmol/L), FeSO4.7H2O (2 mg/L), ZnSO4.7H2O (0.1 mg/L), MnSO4.H2O
(0.03 mg/L), H3BO3 (0.3 mg/L), CoSO4.7H2O (0.24 mg/L), CuSO4.5H2O (0.01 mg/L), NiSO4.7H2O
(0.02 mg/L), NaMoO4.2H2O (0.03 mg/L), Ca(OH)2 (0.5 mg/L), and EDTA (5 mg/L) [10].

2.3. Kinetics of biodegradation of xylene in liquid medium

In liquid tests were carried out successive transfers in order to grow the selected consortium and to
adapt the microorganisms to the contaminant. The consortium was grown in mineral medium to which
xylene was added in different concentrations. The xylene added in transfers is shown in Table 1.

Table 1 – Xylene added to 100 mL inoculum

(*) (*) (*) (*) (*) (*) (*)

Reference #TiXL04 #TiXL08 #TiXL16 #TiXL20 #TiXL24 #TiXL32 #TiXL40

Xylene Volume
4.0 8.0 16.0 20.0 24.0 32.0 40.0
(µL)
Xylene Mass
3.4 6.9 13.8 17.2 20.6 27.5 34.4
(mg)

(*) # stands for the enrichment culture number (1-7); i stands for the transfer number (1, 2 or 3);

The cultures were incubated at 28°C, and shaken at 150 rpm. Xylene concentrations were monitored
by gas chromatography and the incubation period ended when the concentration of xylene in the gas
phase reached 0.5 mg of xylene per liter of air. Figure 2 shows the final aspect of first transfers with
several xylene contaminations.

Figure 2 – Resulting cultures in liquid tests.

Chromatographic determinations were performed in a GC-Shimazu-2010 chromatograph equipped

with a FID and a TRB-5 Teknakroma column (30 m×0.25 mm×0.25 µm). The carrier gas was high-
purity N2; the sample injection was applied in splitless mode. The operating temperatures of injector
and detector were 250ºC, and the column was at 200ºC.
To evaluate the number of colony forming unit (CFU) (Figure 3), 0.5 mL of inoculum was sequentially
diluted in sterile saline solution (0.85% NaCl; W/V),
), spread into LB medium agar and incubated at
28ºC for 3 days [10].

A B C

Figure 3 – Microbial
icrobial consortium: macroscopic view of CFU (A) and a microscopic view (×100) of gram
positive baccillus (B) and a gram negative coccos (C).

2.4. Kinetics of biodegradation of xylene in residual granitic soil samples

The residual granitic soil (Srγ) was previously dried in an oven at 45 º C for 72 hours.

These kinetics studies were first performed in sterilized Erlenmeyer flasks (500 mL) closed by Mininert
valves (Figure 4A). Each
ach flask was prepared with 160 g of soil sample and 40 mL inoculum of the
second transfer of enrichment culture, resulting 200 g of soil with 20% moisture content. Different
xylene supplement was used: 16, 20 and 24 µL. These soil tests sts were designed by SXL16, SXL20 and
SXL24, respectively. Non inoculated media were used as Blank tests for abiotic degradation of xylene.
The tests were replicated
licated for different incubation temperatures: 20 and 26ºC. Xylene concentrations in
the gas phase were monitored by gas chromatography and the the incubation time was also established
at a residual concentration of 0.5 mg xylene per litre of air.

To estimate the number of forming colonies, 10 g of soil was shacked in 100 mL of sterile saline
solution (0.85% NaCl; W/V) for one hour;
hour 0,5 mL of this culture was sequentially diluted in sterile
saline solution and spread into LB medium agar and incubated at 28ºC for 3 days [10].
[

After those preliminary tests, the kinetic studies were made in a column designed for bioventing. It is a
cylindrical stainless steel column with 50 cm height and 10 cm diameter
diameter (Figure 4B).

The column was loaded with 2000 g of residual granitic soil (1600g of dried soil and 200 mL of
T2XL20 plus 200 mL T2XL24 inocula) contaminated with 240 µL L (200 mg) of xylene. These soil tests
were referenced by Col.Xli, were i is the test number. The testss were carried out at room temperature,
monitoring temperatures inside and outside the column. The concentration of xylene in the gas phase
was, like the previous, monitored by gas chromatography and incubation time was dictated by the
value defined as the residual concentration of xylene in the gas phase (0.5 mg of xylene per liter of
air).
 A B

Figure 4 – Tests on residual granitic soil samples. A: Flasks

ks containing contaminated soil used in
kinetics tests. B: A view of the column used in the tests.

3. Results

3.1. – Soil Characterization

The geotechnical characterization tests carried out indicate approximately 7% of clays, 28% of silts,
3
60% of sands and 5% of gravel. Maximum density 1.82 g/cm for a optimum water content of 11.8%,
density 2.68; bulk density 0.955 and porosity 64%.

Physical and chemical characterization revealed pH 4.56, soil conductivity at 25ºC was 66 µS/cm and
organic matter very low (≤ 0.81%).

3.2. Liquid Medium Tests

Liquid medium tests allowed determining

determin the eligible contaminant concentrations for soil testing.
According to the results obtained in the first transfers (Figure 5), concentrations of 0.4 µL of xylene per
mL of inocula (2T1XL40) were not suitable for the bacterial consortium used, since the efficiency of
remediation achieved was very low. The other tested concentrations showed similar remediation times
and high efficiency of remediation, and as such, the consortium was considered appropriate for the
concentration range of 0.04 to 0.32 µL of xylene per mL of inoculum. The higher bacterial growth was
obtained
btained with a concentration of xylene between 0.16 and 0. 24 µL L per mL of inoculum.

Figure 5 – Liquid medium test results. Left: Wide range of xylene concentration. Right: Narrow
range of xylene concentration.
At the end of remediation time the biomass was quantified by counting CFU, the results are plotted in
Figure 6, where CFU/mL is compared to xylene volume used in different transfer cultures. This may
indicate that 0.4 µL
L of xylene per mL of inoculum becomes toxic for microorganisms.

The results verify that cultures with higher content in xylene have very low bacterial.
bacterial In the other
situations, bacterial growth increased with the amount of xylene (substrate) added to the culture.

Figure 6 – Biomass versus xylene content.

Analyzing the remediation efficiency of different transfers of the same enrichment culture (Figure 7) it
can be concluded that the efficiency of remediation achieved are identical. However, remediation time
is different, being optimal for the second transfer. Based on these results the second transfer was
chosen as inoculum in subsequent soil tests.

Figure 7 – Liquid medium test

t results: different transfers (T1, T2 and T3)) of same enrichment
culture (4).
 3.3. – Residual Granitic Soil Tests

The bioremediation tests in soil using 16, 20 and 24 µL

L of xylene showed identical results.

Figure 8 presents the soil with different incubation temperatures,

temperature , showing that incubation temperature
does not substantially affect the remediation efficiency.
efficiency Itt should be noted that the higher temperature
(26ºC) allows a faster startup of biodegradation, while there is a greater initial period of acclimation to
lower temperatures.

Figure 8 – Residual
sidual granitic soil - flask tests: different incubation temperatures for the same xylene
concentration.

Based on these results, tests were then performed in a column at room temperature. Furthermore, the
contamination grade selected was 200 mg of xylene per kg of soil. The results for the tests in two
columns (Figure 9) indicated remediation efficiency identical to those obtained in flasks with
remediation time varying from 7 to 13 days.

Figure 9 – Residual granitic soil - column tests

 3.4. Final Considerations

The comparison between some e results obtained in two soil

soil-scale tests and in liquid tests is presented
in Figure 10. It shows that the time remediation was longer in soil tests, but the final remediation
efficiency was the same in all tests. Therefore, the he consortium developed proved
pr to be able to
biodegrade xylene in both media and scale tested.

Figure 10 – Comparison between column tests (ColXL01

( and ColXL02),
), flasks tests with soil
(SXL20_1
SXL20_1) and flasks tests with medium (4T2XL24).

Based on these results, it is now possible, to test this bacterial consortium in bioventing remediation.

4. Conclusions

The consortium developed proved to be able to degrade

degrad xylene in both media and scale tested.

Concentrations of 0.4 µLL of xylene per mL of inocula

inocul (2T1XL40) were not suitable for the bacterial
consortium used, since the efficiency of remediation achieved and bacterial growth were very low.

The studies were carried out in the range of concentrations of the contaminant adequate to bioventing
technology application.

The results show convincing adequacy of the bacterial consortium

consortium at this concentration range,
promising good results in full scale bioventing.

5. References

[1] Fiúza, A., “Reabilitação de solos e aquíferos contaminados”, RSA - Support text for an
undergraduate Course, 2009, FEUP.
FEUP

[2] EPA, “How to

o evaluate alternative cleanup technologies for underground storage tank sites: a
guide for corrective action plan reviewers”
reviewers EPA 510-R-04-002,002, 20th April 2010,
http://www.epa.gov/oust/pubs/tums.htm.
[3] Sui, H., Li, X., Jiang, B.: “Benzene, toluene and p-xylene interactions and the role of microbial
communities in remediation using bioventing”, The Canadian Journal of Chemical Engineering 83,
310-315, April 2005.

[4] Fritsche, W., Hofrichter, M. “6.Aerobic degradation by microorganisms”, A Multi-volume

comprehensive treatise, Biotechnology Second, Completely Revised Edition, WILEY-VCH, 2000.

[5] Especificação LNEC E 195/1966 – “Preparação por via seca de amostras para ensaios de
identificação”. LNEC, Lisboa, 1966.

[6] Especificação LNEC E 196/1966 – “Análise granulométrica”. LNEC, Lisboa, 1966.

[7] Especificação LNEC E 197/1966 – “Ensaio de compactação”. LNEC, Lisboa, 1966.

[8] Norma Portuguesa NP83/1965 – “Determinação da densidade das partículas”, Lisboa, 1965.

[9] Hesse, P.R.: “A textbook of soil chemical analysis”, Chemical Publishig CO, 1972.

[10] Vila, M.C., Nunes, O. P., Fiúza, A.: “A model of biodegradation of crude oil in soils”,
Proceedings of the First Bioremediation Conference, Chania, Greece (2001) , pp 1-4.