ORIGINAL PAPER

Microbial communities and biodegradation in lab-scaleBTEX-contaminated groundwater remediation usingan oxygen-releasing reactive barrier

Chi-Wen Lin Æ Li-Hsuan Chen Æ Yet-Pole I ÆChi-Yung Lai

Received: 5 April 2009 / Accepted: 24 May 2009 / Published online: 10 June 2009

� Springer-Verlag 2009

Abstract To remediate benzene, toluene, ethylbenzene

and xylene (BTEX) -contaminated groundwater, a bio-

treatment process including biostimulation and bioaug-

mentation was simulated using oxygen-releasing reactive

barriers (ORRB) and water with added BTEX in a lab-scale

system. The results showed that the capability for BTEX

removal decreases in the order of benzene, toluene,

p-xylene, ethylbenzene for both added-nitrogen and no-

added-nitrogen under BTEX concentrations at 30 mg l-1.

The removal efficiencies in ORRB systems were higher in

the nitrogen-added condition for biostimulation compared

with the no-nitrogen-added condition; moreover, an

increased pattern for removal was observed during the

bioaugmentation process. The oxygen content was found to

be inversely proportional to the distance from the ORRB,

as evidenced by observing that the average bacteria den-

sities were two orders higher when located at 15 cm

compared with 30 cm from the ORRB. The microbial

community structure was similar in both cases of added-

nitrogen and the no-added-nitrogen conditions.

Keywords Biostimulation � Bioaugmentation �Microbial community structure �Oxygen-releasing reactive barrier (ORRB)

Introduction

BTEX (benzene, toluene, ethylbenzene and xylene) has

been widely used in the petrochemical industry, printing

and laminating facilities, foundries, electronics, and paint

manufacturing plants. BTEX is frequently found at haz-

ardous waste sites [1]. Tank leaks or ruptured pipelines

cause BTEX-polluted soil and groundwater. In recent

years, several studies on BTEX treatment have been

implemented to remediate BTEX-contaminated sites

[2, 3]. In situ bioremediation has been applied because of

its capabilities for providing bacteria with environmen-

tally appropriate substances such as oxygen and nitrogen

[4, 5]. A permeable reactive barrier (PRB) is technolog-

ically suitable for contaminated groundwater treatment.

When a PRB is used, contaminated groundwater flows

through a vertically reactive material in which contami-

nants are physically, chemically or biologically degraded

[6]. For groundwater in situ remediation, it should

be more suitable when the technology is applied for

increasing aerobic metabolism than for anaerobic. There-

fore, using an oxygen-releasing reactive barrier (ORRB)

both renders the environmental condition aerobic and

overcomes the reduction state caused by an anaerobic

condition, thereby obtaining higher degradation effi-

ciency. Oxygen-releasing compounds (ORC), a mixture

of CaO2 or MgO2, cement, sand and other materials of

certain proportions have all been researched and applied

to contaminated groundwater studies and remediation

projects [7–9]. The use of such substances (i.e., ORC) is

C.-W. Lin (&) � Y.-P. I

Department of Safety, Health and Environmental Engineering,

National Yunlin University of Science and Technology, 123

University Road, Sect. 3, Douliou, Yunlin 64002, Taiwan, ROC

e-mail: linwen@yuntech.edu.tw

L.-H. Chen

Department of Environmental Engineering, Da-Yeh University,

Dacun, Changhua, Taiwan, ROC

C.-Y. Lai

Department of Biology, National Changhua University

of Education, Changhua, Taiwan, ROC

123

Bioprocess Biosyst Eng (2010) 33:383–391

DOI 10.1007/s00449-009-0336-7

extendible for 6 months or even 1 year, having shown

more economic benefits than pump and treat or air

stripping (50–70% cheaper) [10].

Studies of biostimulation (added nutrients, electron

donor or acceptor) and bioaugmentation (added degrad-

ing strains) applications have also been reported. For

example, nitrogen, phosphorus and oil-degrading bacteria

have been added to petroleum-hydrocarbons contami-

nated soil. The results showed that the addition of such

bacteria enhances the removal of hydrocarbons; more-

over, adding nitrogen and phosphorus stimulates the

microbial growth [5, 11, 12]. Hristova et al. [4] and

Salanitro et al. [13] used bioventing along with the

addition of MTBE-degrading bacteria to demonstrate that

higher efficiency can be achieved by combing the two

methods than by using a single one. Oxygen-releasing

compounds have been used to provide oxygen to stim-

ulate the microbial growth, for which NaNO3 and

(NH4)2SO4 can be used as nutrients. When there is a

lack of oxygen, NO3-and SO4

2- can also function as

electronic receptors [14, 15].

The factors influencing the effectiveness of bioremedi-

ation are closely related to microbial distribution and

growth conditions [16]. Consequently, it is important to

investigate the relationships between the factors including

types of contaminants and environmental conditions, and

microbial communities. Microbial data collected by using

molecular biotechnology techniques are more useful than

traditional ones. SSCP (single-strand-conformation poly-

morphism), a low-cost and highly sensitive method [17,

18], has been used in recent years to detect microbial

diversity [19–21].

ORCs have also been used for recovery at sites con-

taminated by some organic pollutants. For example, an

ORC method was applied to 50 monitoring wells to treat

MTBE pollution in a fuel tank leakage of contaminated

water [22]. In a lab-scale system, a string of double-wall

bioreactor systems was tested to deal with tetrachloro-

ethylene pollutants [7]. In another study, bioaugmenta-

tion and oxygen used were focused on accelerating the

biodegradation process [8]. Microorganisms play an

important role in the degradation of pollutants; however,

microorganisms easily change with the environmental

factors. Therefore, this study employed a polymerase

chain reaction and single-strand conformation polymor-

phism (PCR-SSCP) technique to analyze changes in

microbial population dynamics and to overcome the

limits of traditional quantitative plating methods in

which microorganism analyses cannot fully reveal the

species and the resulting microbial community

composition in the environment. The study also ana-

lyzed the effectiveness of ORC, biostimulation and

bioaugmentation.

Materials and methods

Microorganisms

Mixed cultures were obtained from two sources, one from

a gasoline-contaminated groundwater site, and the other

from industrial wastewater treatment sludge in Mailiao

Industrial Park, Taiwan. A mixture of the two sources was

further defined as ‘in situ microorganisms’. BTEX-

degrading strains were isolated from laboratories in which

the incubation occurred. GenBank BLAST analysis of 16S

rDNA genes (http://www.ncbi.nlm.nih.gov), based on the

sequence of a *1,400-bp fragment has previously revealed

that the accession numbers, percentage of similarities and

E values for the matching score (indicated in parentheses)

are Pseudomonas sp. (DQ211692, 100%, 0.0), Pseudomo-

nas sp. (AF065166, 99%, 0.0), Pseudomonas putida

(AY686638, 100%, 0.0), and Pseudomonas sp. (DQ124297,

99%, 0.0), respectively.

Degradation of BTEX in an ORRB system

An oxygen-releasing reactive barrier (ORRB) system was

divided into three parts: oxygen-releasing reaction wall, a

water supply and collection systems, and a temperature

control system, as shown in Fig. 1. Table 1 lists the system

specifications. The inlet concentrations of benzene, tolu-

ene, ethylbenzene, p-xylene were maintained at 25–36, 26–

36, 26–39, and 27–38 mg l-1, respectively. The inflow rate

was set at 3.45 ml min-1 with 50 cm of flow velocity per

day. The ORRB reactor was maintained at a temperature of

23–25 �C by using temperature-controlled circulating

water outside the reactor. The ORRB reactor was filled

with approximately 25-kg Ottawa quartz sands (E-315,

GEOTEST, USA). The reactive barrier in ORRB reactor

(Fig. 1) was filled with 800 g of ORC [23] consisting of

cement, sand, 40% CaO2, KH2PO4, K2HPO4, NaNO3, and

H2O. The reactor was completely closed; thus, loss of

BTEX in the analytical process was restricted.

Two systems (A and B) were tested in this study. System

A was supported by a nitrogen nutrient and filled with an

ORC consisting of 40% CaO2 and 11% NaNO3. System B

was filled with an ORC composed of only 40% CaO2.. Both

systems were run with the same phases, the first phase being

biostimulated by the added ORC and the implantation of

3,000 ml in situ microorganisms (4.62 9 108 CFU ml-1).

The second phase was a continuation of the first with bio-

augmentation (added of BTEX-degrading strains at sites #1,

#2, and #3; 200 ml per well; 8.49 9 109 CFU ml-1). After

a 15- to 20-day start-up period, the system remained nor-

mally stable with a difference in removal efficiency of less

than 5% (the first phase), which continued in the second

phase.

384 Bioprocess Biosyst Eng (2010) 33:383–391

123

Pollutants and water analysis

A purge water procedure was performed for effectively

eliminating the production of purge water when obtaining a

groundwater sample from a monitoring well. According to

the procedure, the equivalent volumes of water of 2–3

times the wells’ volume were first removed and discarded

at sites #4-a, #4-b, and #4-c using needles and a peristaltic

pump. Subsequently, 10-ml water samples were collected

from the wells, to await water quality analysis for dissolved

oxygen (DO), oxidation reduction potential (ORP), chem-

ical oxygen demand (COD), pH and SSCP. Moreover, 3 ml

of samples were taken by the needles, after which 1 ml of a

subsample was sealed in a 3-ml glass bottle to await further

analysis.

Key physical and chemical properties of BTEX include

Henry’s law coefficient, vapor pressure, and water solu-

bility. The change of pollutant concentrations in the gas

and liquid phases in each bottle can be related using

Henry’s law. Because Henry’s law coefficients of benzene,

toluene, ethylbenzene and xylene are 0.22, 0.27, 0.32 and

0.20 at 25 �C, respectively, show BTEX compounds are

volatile organic compounds, mass transfer rates between

the gas and aqueous phases of BTEX are rapid, and gas

concentrations approach equilibrium with the liquid phase.

Therefore, use of gas-phase measurements for biodegra-

dation experiments satisfactorily captures the pollutant

concentrations in the liquid. Gaseous samples of BTEX

obtained from the headspace of each 3-ml glass bottle were

then injected onto GC-FID (gas chromatograph, model

GC-14B, Shimadzu, Japan) by 250-ll gastight syringes

equipped with Teflon Mininert valve fittings. Concentra-

tions of BTEX were quantified against primary standard

curves.

Similarly, water samples from sites #1 to #4 were col-

lected and analyzed for COD, DO, ORP, pH, NO3--N and

colony-forming units (CFU). Each volume of 15-ml sample

taken by the needles was used for water quality analysis,

and an additional 1.5 ml water sample was collected by a

microcentrifuge tube and preserved at -20 �C for DNA

extraction.

DNA extraction, PCR and SSCP gel electrophoresis

DNA was extracted by using an improvement on a bead-

beating method developed by Stach et al. [19]. A ground-

water sample (200 ll) was mixed with 0.8 g of 0.106-mm

glass beads (Biospec Products, 11079101), 600 ll of

phenol/chloroform/isoamyl alcohol (25:24:1), and 200 ll

of an air-excluding disrupting buffer (50 mM NaCl,

50 mM Tri–HCl, pH 8 and 5% SDS) in a 1.5-ml screw-cap

microcentrifuge tube. The microbial communities were

analyzed by using the PCR-SSCP method described by Lee

et al. [17] and Schwieger and Tebbe [18]. Region V3 of the

16S rDNA, corresponding to nucleotide positions 334–514

of the E. coli gene, was amplified with the primers EUB1

(50-CAGACTCCTACGGGAGG CAGCAG-30) and UNV2

(50-GTATTACCGCGGC TGCTGGCAC-30). A Hoefer

SE600 vertical gel electrophoresis apparatus was used for

Fig. 1 Schematic diagram of an

oxygen-releasing reactive

barrier system

Table 1 Summary of oxygen-releasing reactive barrier system

specifications

Items Specification

Material Crystalloid PVC

Total volume 15.9 L (530 9 150 9 200 mm)

Filler Quartz sand

Oxygen filler Oxygen-releasing compounds

(ORC)

Inside-pore volume 5.1 L

Porosity 0.338

Water-surface slope 0.0099 m m-1

Hydraulic conductivity 2.9 9 10-2 cm s-1

Circulating water volume

(outside)

6.3 L

Temperature control (outside) 20–25 �C

Bioprocess Biosyst Eng (2010) 33:383–391 385

123

SSCP analysis in 10% polyacrylamide gel for 6 h at a

constant voltage of 300 V. The gel temperature was

maintained at 4 �C by using a circulating water bath.

Details of this procedure can be found in the protocol

described by Lin et al. [16].

Statistical comparison of SSCP pattern

The relative positions of the DNA bands in the SSCP gels

were analyzed by using LabWork software. Similarities

between microbial groups were calculated as Dice indices

according to procedures appearing in previous reports [16,

24]. Dendrograms were calculated by using a clustering

algorithm of a UPGMA using cluster analysis of similarity

indices, constructed by NTSYSpc Version 2.1e software

(Exeter Software, USA).

Results and discussion

Preliminary tests for ORC, DO and nitrogen effects

Figure 2 shows the results of background tests with DO

distribution sampled from monitoring wells in an ORRB

system during a 6-day operation. Na2SO3 was added to the

inflow water at 80 mg l-1 to ensure a low DO of inflow

(0–0.5 mg l-1). The average DO concentration at site #1

(-10 cm) was as much as 4 mg l-1, a result caused by the

higher diffusion rate of oxygen released from the ORC than

by the flow rate of the groundwater, which further provided

an elevated DO near the upstream region at site #1. The

average DO was detected in a range of 7.51–8.27 mg l-1 in

the downstream region at sites #2 (10 cm), #3 (15 cm) and

#4 (30 cm), showing that oxygen escaping into the atmo-

sphere was negligible. A column containing ORC was

tested, and thereby indicating that oxygen was stably

released, having persisted at least 35 days (data not

shown). A large quantity of DO of 8 mg l-1, approxi-

mately, was found around this column, thus indicating that

the ORCs create an aerobic circumstance by releasing

oxygen, and DO functions as an electron acceptor in the

aerobic metabolism of bacteria in an ORRB system for

BTEX biodegradation, the results being consistent with

other studies [25, 26].

Figure 2 also depicts the NO3--N concentration distri-

butions for the ORRB system during the 6-day operation.

At site #1, the average concentration of NO3--N was below

1 mg l-1, while the values downstream ranged between

100 and 200 mg l-1. The low concentration of NO3--N

detected at site #1 revealed that the diffusion rate was not

as high as that of the oxygen, the release rate of which was

higher than the flow rate. The detected NO3--N concen-

tration downstream was approximately 1% of the designed

concentration in the ORC, thereby indicating that the

nitrogen source was successfully released.

BTEX removal efficiency

This study was implemented to test the efficiency of an

ORRB system and the biodegradation of pollutants. A

comparison of systems A and B indicated higher removal

efficiency in the first phase, wherein nitrate was added

because this nutrient enhances BTEX-degrading bacteria

activities under both aerobic and anaerobic conditions, as

illustrated in Fig. 3. Nitrogen is one of the most essential

elements for living, and nitrate is an electron acceptor.

These findings are consistent with other published reports

[2, 27]. In the first-phase experiment (biostimulation) for

system A, DO functioned as an electron acceptor; whereas,

NO3--N can be assumed to promote the capacity of the

biodegradation [2]. Therefore, the removal efficiency for

benzene, toluene, ethylbenzene and p-xylene in system A

was greater than in system B by 14.3, 22.6, 24.9, and

27.3%, respectively. In the second phase (bioaugmenta-

tion), the overall removal efficiency of system A was still

slightly higher than that of system B. In contrast, a note-

worthy improvement in removal efficiency by 16, 14, 20,

and 28% for BTEX, respectively, was observed for system

B as a result of the addition of BTEX-degrading bacteria.

These observations are in agreement with other recent

reports [28, 29]. The improvement in removal efficiency in

system A just increased by 15, 11, 5, and 1% for BTEX,

respectively. The limited enhancement in system A was

believed to be due to the fact that nitrogen addition had

only a small effect in phase 2.

Water sample analysis

In this study, ORP, COD, BTEX, DO, pH and biomass

were used as biological indicators for the bioremediation

process. The ORP values at the monitoring wells for sys-

tems A and B ranged between 100 and 200 mV, thereby

indicating that both systems were aerobic throughout. High

pH values (8–10) were observed during the first 6 days of

Fig. 2 Oxygen distribution and NO3--N concentration in an ORRB

system (background tests) at four sites

386 Bioprocess Biosyst Eng (2010) 33:383–391

123

testing; whereas, neutral values (6.8–7.5) were consistently

detected thereafter. Figure 4 plots the COD variation in the

systems (A and B), showing different values between the

inflow and site #4 (30 cm downstream). The COD values

were unstable during the first 10 days, but subsequently

decreased from 143 to 38 (system A) and 48 mg l-1

(system B); therefore, the removal efficiencies increased

73.4% for system A and 66.1% for system B. In the

second-phase experiment, the removal efficiency increased

during the first several days but remained in the range from

75 to 85% for system A and 65 to 75% for system B.

(A)

(B)

p

Fig. 3 Average removal efficiencies in systems A (with added

nitrogen) and B (without nitrogen). ‘biostimulation’ applied in the

first phase (average removal efficiencies during days 10–20), and both

‘biostimulation and bioaugmentation’ applied in the second phase

(average removal efficiencies during days 25–45)

-1

(A)

(B)

-1

Fig. 4 Variation in COD and removal efficiency in systems A (with

added nitrogen) and B (without nitrogen)

-1-1

(A)

(B)

Fig. 5 Variation in DO in systems A (with added nitrogen) and B(without nitrogen)

-1

(A)

(B)

-1

Fig. 6 Variation in cell density in systems A (with added nitrogen)

and B (without nitrogen)

Bioprocess Biosyst Eng (2010) 33:383–391 387

123

Similarities were observed when comparing the COD

values and the BTEX concentrations. This finding indicates

that BTEX can be effectively decomposed into CO2 and

water or other substances (biomass and other organic

compounds); moreover, the changes in COD concentration

were obvious evidences for BTEX degradation.

Figure 5 shows that the amount of DO was quite high at

the beginning of the first phase, an observation interpreted

as being due to a rapid reaction of ORC with water, thereby

generating a great quantity of oxygen, which still had not

been depleted by bacterial utilization at this time. After the

first 10 days of operation, the DO was significantly reduced

(a)

(b)

Fig. 7 Cluster analysis of

SSCP profiles of microcosms in

system A at site #3: a SSCP

fingerprint, b cluster analysis

388 Bioprocess Biosyst Eng (2010) 33:383–391

123

and reached a steady state; the highest being found at site

#2, followed by site #3, and the lowest at sites #4 and #1.

Therefore, the decrease in DO from upstream to down-

stream can be attributed to the utilization of oxygen to the

bacteria, thereby increasing the bacterial population.

Figure 6 depicts the changes in cell density in systems A

(with added nitrogen) and B (without nitrogen). At

monitoring site #3, this value increased from 4.1 9 106

(day 5) to 1.3 9 107 CFU ml-1 (day 19) in both systems.

The remaining wells exhibited a similar pattern. At the

beginning of the second phase in system A, the DO tended

to decrease from 1.77 (day 21) to 0.86 mg l-1 (day 26) and

subsequently stabilize between approximately 0.75 and

1.13 mg l-1 (the value at site #4 was even lower at

(a)

(b)

Fig. 8 Cluster analysis of

SSCP profiles of microcosms in

system A at site #4: a SSCP

fingerprint, b cluster analysis

Bioprocess Biosyst Eng (2010) 33:383–391 389

123

0.11–0.35 mg l-1). Although the oxygen at site #2 was

continuously released from ORC, it was rapidly consumed

by a significant growth of bacteria, thus resulting in a

constant value of DO at 2.03–3.15 mg l-1.

Figure 6 also indicates that the CFU value at site #3

increased to 1.5 9 108 CFU ml-1 in the second phase, a

result caused by the addition of BTEX-degrading bacteria.

However, the microbial population was lower at site #4

near the end of the flow, indicating a possible washout of

microorganisms. Figure 6 also shows a higher CFU value

from days 3–45—particularly in days 10–20—in system A

in comparison with B because nitrogen, which can be

required for biomass construction, was added in only sys-

tem A.

Variations within microbial communities in monitoring

wells

PCR-SSCP was use to characterize the community distri-

bution in both systems at the monitoring wells (sites #3 and

#4) from the two-phase study. The removal efficiency, DO

and CFU were compared to assess the feasibility of this

method as well as the relationship between BTEX degra-

dation and the microbial growth or declination.

Figure 7 shows a UPGMA cluster analysis of the SSCP

profiles of microcosms at site #3 in system A. Three

biomarkers with highly similar sequences were used,

including Pseudomonas sp. (DQ211692), Pseudomonas

sp. (AF065166) and Pseudomonas putida (AY686638);

therefore, the three observed bands (c, d and e) were quite

similar. However, the Pseudomonas sp. (DQ124297) was

screened from a source different from that of the bacteria

mentioned earlier, and its bands (a, b, d and e) were slightly

different. The relative similarities indicated in Fig. 7b can

be classified into two phases for system A: (1) days 0, 5, 9,

12 and 19; and (2) days BA (bioaugmentation at day 20),

and days 22, 25, 28, 35, 40 and 45. Among the different

phase-tests, the diversity in the microbial community was

the greatest; therefore, and the least similarity (31%) was

exhibited. A low correlation between the groups for days 0

and 5 (about 0.65) was also indicated. The reasons appear

to be that the BTEX and COD removal efficiency was

gradually enhanced after bioaugmentation (10–20 days, as

plotted in Fig. 4A), and that the DO consumed by the

microorganisms resulted in an increase in the bacterial

population (as plotted in Fig. 5A). These possible reasons

led to a slight change in the microbial structure at site #3,

thereby tending to form the predominant bacteria as in the

first 19 days of this study. The results obtained from sys-

tem B were similar to those from A (data not shown). On

the basis of the analysis illustrated in Fig. 7a, the BA had a

correlation of 0.66 with the biomarker. The system con-

ditions gradually stabilized, thus becoming suitable for the

growth of bacteria. Days 22 and 25 paired had a similarity

of 0.66; days 28 and 35 had 1.0; days 40 and 45 had 0.89.

Since the supply of oxygen and substrate was adequate at

site #3, this site was more diversified than sites #1 and #4.

The results obtained from system B were similar to those

from A.

Figure 8 depicts a cluster analysis of the SSCP profiles

of the microcosms in system A at site #4. In both sys-

tems, the microcosms on day 22 and the marker were

highly similar (0.9 in A and 0.67 in B); whereas, on day

28, the similarities were reduced to 0.53 and 0.56 in

systems A and B, respectively. Because site #4 was

located near the outlet region and the supply of DO was

almost depleted at site #3, the bacterial structure at site

#4, including both aerobic and anaerobic bacteria, was

more complex than at site #3.

Conclusion

This study has demonstrated the use of ORRB to treat

BTEX-polluted groundwater. Both biostimulation and

bioaugmentation can accelerate the microbial activity and

degradation of pollutants. Moreover, the highest CFU value

and microbial diversity were found 15 cm downstream.

Both removal effects and stable CFUs were gradually

achieved. PCR-SSCP can be effectively used to explain the

changes in microbial structure, when CFU and environ-

mental information are provided. Determining the rela-

tionships among the BTEX removal efficiency, COD, DO,

bacteria densities and the microbial community structure

are the capable tools to assess the effectiveness of using

ORRB in BTEX-contaminated groundwater.

Acknowledgments This study was funded by the National Science

Council of the Republic of China under contract NO. NSC 96-2221-

E-224-093-MY3. The authors also wish to express their appreciation

to Dr. Cheryl J. Rutledge for her editorial assistance.

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