Development of an attached-growth process for the bioremediation of trichloroethylene- and
1,1,2,2-tetrachloroethane-contaminated groundwater
Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco Doria, Stefano
Lei, Valentina Spaggiari, Nasrin Tavanaie, Florin A. Potra, Roberta Ciavarelli,
Davide Pinelli, Serena Fraraccio, Giulio Zanaroli, Fabio Fava.
DICAM, University of Bologna
Via Terracini 28, Bologna, Italy
Acque sotterranee: protezione dai rischi igienico sanitari e/o tutela delle risorse
RIMINI, 8 NOVEMBRE 2013
Chlorinated aliphatic hydrocarbons (CAHs)
CAH-contaminated waters are usually treated by means of physical–chemical methods
(stripping and adsorption) which simply transfer the contaminants to a different matrix.
On the other hand, several studies have indicated that the biological
degradation/transformation of CAH can occur under aerobic and anaerobic conditions,
offering the possibility to transform several CAHs into non-toxic, easily biodegradable compounds.
Trichloroethylene (TCE) 1,1,2,2-Tetrachloroethane (TeCA)
Chlorinated aliphatic hydrocarbons (CAHs) represent a widespread cause of water contamination. Due to their toxicity and the demonstrated cancerogenicity of some of
them, subsurface contamination by CAH is considered particularly serious and dangerous.
Aerobic cometabolic biodegradation of CAHs
Low-chlorinated CAH (1, 2 or even 3 chlorine atoms) are in general more easily degraded by
means of aerobic (oxidation) processes, which offer the possibility to achieve a complete dechlorination of the parent compounds at high degradation rates. However, most CAH
aerobic degradation processes occur by means of a cometabolic pathway
Thus, it is required to supply a proper growth substrate (an aliphatic or aromatic hydrocarbon), that provides energy and induces the synthesis of enzymes that catalyze CAH
biodegradation.
MONO-OXYGENASE
CAH
CC
ClCl
ClH
O
Epoxide
AlcoholsOrganic acids
O2 H2O
Growth substrate CO2 + H2O + energy
Alvarez-Cohen and Speitel Jr. (2011) Biodegradation 12:105-126.
Suttinun et al. (2013) Rev Environ Sci Biotechnol 12:99-114.
Implementation of CAH aerobic cometabolic
processes
The stimulation of in-situ aerobic cometabolic processes presents significant challenges
related to the risk of a complete consumption of the supplied growth substrate within a
short distance from the injection wells and to the possible clogging of the aquifer porosity.
On the contrary, the on-site implementation of aerobic cometabolic processes presents
quite interesting opportunities, in particular when the need to implement a hydraulic
barrier leads to the choice of a pump and treat remediation approach.
Among the possible bioreactor solutions, packed-bed bioreactors (PBRs) present specific advantages over bioreactors
with suspended cells.
Gandhi et al. (2002) Wat Resour Res 38:11-1–11-18.
Aim of the research
To develop an on-site aerobic cometabolic bioremediation process based on a
packed bed bioreactor (PBRs) for an actual site ground water contaminated by
Trichloroethylene (TCE) and 1,1,2,2-Tetrachloroethane (TeCA) located in Northern Italy.
The site is articulated in a shallow and a confined aquifer contaminated mainly by TCE (0.04-5.8 mg L-1)
1. to obtain and characterize an effective CAH-degrading microbial consortium from the site’s indigenous biomass and select the best growth substrate for the AC process;
Specific aims of this work
2. to select the best carrier for the PBR process;
3. to develop the PBR continuous-flow process with pulsed supply of substrate and
oxygen;
4. to scale-up the PBR continuous-flow process to a 31-L plant.
This research is part of the EU FP7 research program “Microorganism and enzyme
Immobilization: NOvel Techniques and Approaches for Upgraded Remediation of Underground-, wastewater and Soil” (MINOTAURUS).
1) SELECTION OF SUBSTRATE/MICROBIAL
CONSORTIUM
4 groundwater samples (n. 1 to 4) collected from different zones of the aquifer and a
mixture of the same groundwaters (n. 5) were incubated in the presence of 4 candidate
growth substrates at 30°C for 60 days.
The 20 enriched consortia were
compared in terms of biodegradation capabilities
(normalized net biodegradation
rates) and structure of the microbial community (DGGE).
M1 M2 M3 M4 M5
B1 B2 B3 B4 B5
PE1 PE2 PE3 PE4 PE5
PR1 PR2 PR3 PR4 PR5
Methane (M)
Butane (B)
Pentane (PE)
Propane (PR)
Growndwater samples
1 2 3 4 5
Based on its high k1,TCE (96 L
gprotein-1 d-1), the consortium enriched on butane from
groundwater n. 4 (i.e., B4),
was selected as the best performing one and further
characterized.
2) SELECTION OF THE BIOFILM CARRIER
4 types of porous biofilm carriers were tested:
Biomax:• Porosity: 60%• Density: 0.66 kg/L• Ceramic
Cerambios:• Porosity: 74%• Density: 0.66 kg/L• Ceramic
Biomech:• Porosity: 64%• Density: 0.68 kg/L• Ceramic
Biopearl:• Porosity: 58%• Density: 0.95 kg/L• Sintered glass
Consortium B4 was subcultured at 30°C and 15°C in 50 mL of sterile groundwater + 60 mL of
carriers, with butane (2 mg/L), oxygen (8 mg/L) and TCE (1 mg/L) pulses. Liquid phase was
periodically replaced with sterile water to remove freely suspended cells.
BIOPEARLBIOMECHBIOMAXCERAMBIOS
After 8 spikes, carriers were washed with sterile
physiological solution. Normalized degradation rates
and attached biomass concentrations were measured and the biofilms characterized.
0.00
0.04
0.08
0.12
0.16
Biomax Biomech Biopearl Cerambios
30°C 15 °C
0.0
0.2
0.4
0.6
0.8
Biomax Biomech Biopearl Cerambios
30 °C 15 °C
(d-1
)
(gp
rote
in
Lb
iore
acto
r-1)
Biomax was characterized by the highest values of attached cells concentration at both temperatures.
Biomax also exhibited the highest normalized TCE degradation rate at 15°C and at 30°C (along with Biomech).
Frascari et al. (2013) Biodegradation in press DOI: 10.1007 / s10532-013-9664-z.
2) SELECTION OF THE BIOFILM CARRIER (continued)
Biomax was thus selected as the best performing immobilization carrier.
Attached cell concentration TCE normalized degradation rate
Kinetic model: TCE, 30°C
0 2 4 6 8 10
-0,02
0
0,02
0,04
0,06
0,08
0,1
Initial TCE concentration (mg/L)
Initial specific TCE depletion rate (g/g prot./d)
� Model: competitive inhibition
� Butane concentration: 1.5 mg L-1
qmax, TCE, attached cells = 0.021 ± 0.001 g gprotein-1 d-1
Ks, TCE, attached cells = 1.10 ± 0.07 mg L-1
TCE
TCEBcompI
TCEs
TCETCE
CK
CK
Cqq
+
+
⋅=
−
⋅
,,
B
,
max,
1
Ki,comp,B-TCE, attached cells = 0.778 ± 0.23 mg L-1
KINETIC ANALYSIS OF TCE BIODEGRADATION BY BIOMAX-ATTACHED CELLS OF THE SELECTED CONSORTIUM, WITH OR
WITHOUT BUTANE INHIBITION
Goals of the pulsed feed of substrate and oxygen:
� to reduce substrate inhibition on the CAH biodegradation rate
� to extend the bioreactive zone to the entire reactor length, with a homogeneous
biomass distribution
3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED
SUPPLY OF SUBSTRATE AND OXYGEN
0
5
10
15
20
0 6 12 18 24
Time (hours)
Co
nce
ntr
atio
n (
mg
/L)
Oxygen
Growth substrate
Concentration vs. time at the bioreactor inlet
The overlapping of oxygen and growth substrate , and therefore the uptake of substrate,
occurs in 1 or more narrow bioreactor zones, that shift with groundwater flow and that are
characterized by low oxygen and growth substrate concentrations
� low substrate uptake rate � long bioreactive zone
� substrate inhibition on TCE cometabolism limited to the zones where, at each instant,
substrate is present
0
5
10
15
20
0 0.5 1 1.5 2
Co
nce
ntr
atio
n (
mg
/L)
Concentration vs. space along the bioreactor
Oxygen
Growth
substrate
Length of active aquifer or column height (m)
Supply of alternated pulses of oxygen and growth substrate
3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED
SUPPLY OF SUBSTRATE AND OXYGEN (CONTINUED)
A 1 L column plant was set-up, filled with the Biomax carrier and inoculated with
consortium B4 (30°C). Passive biomass immobilization was performed in batch for 48 h. The
plant was then fed in continuous mode with alternate pulses of butane (25 mg L-1, pulse length 7.2 h) and oxygen (21 mg L-1, pulse length 16.8 h) and a constant TCE
concentration (1.2 mg L-1) for ̴100 days.
0%
10%
20%
30%
40%
50%
60%
40 50 60 70 80 90 100 110
TCE removal
Time (d)
TCE REMOVAL VERSUS TIME
- The degree of mineralization to Cl- of the
organic chlorine was equal as an average to 90%
PB
R
n.1
–
C
arr
ier:
Ce
ram
bio
sTCE
satura
ted
soluti
on
Control
unit
Oxygen
enrichedwater
Butane enrichedwater
3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED SUPPLY OF
SUBSTRATE AND OXYGEN (CONTINUED)
At the end of operation under continuous flow, carriers were collected from the bottom (inlet,
1), middle (2) and top (outlet, 3) sections of the column, washed in sterile physiological solution, and subjected to the evaluation of:
1
2
3
r/c (1/d)
X (mgprot. /L)
k1 (L/d/mg)
0.15 ± 0.03
28 ± 11 0.06 ± 0.03
0.17 ± 0.06
27 ± 11 0.06 ± 0.03
0.24 ± 0.08
44 ± 18 0.05 ± 0.03
- the TCE
normalized
degradation rate
- the amount
of attached
biomass1 2 3
- the community structure (DGGE
analysis)
- the TCE 1st
order degr.
constant
3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED SUPPLY OF
SUBSTRATE AND OXYGEN (CONTINUED)
L
reactor
Lmol
L
vD
Lv
dispersiondiffusion
convectionPe
αα≅
⋅+
⋅=
+=
int
int
Pe
cle
t
nu
mb
er
-
+Concentration VS time
INLET profile
butane oxygen
Time (d)
Conc. (m
g L
-
1)
0
10
20
30
40
50
0 1 2 3
Substrate and oxygen
pulsing injection feeding
� The substrate pulses remain
separate, along the reactor
� More space for contaminant
degradation without substrate
inhibition
� Low butane/oxygen
overlapping � low average
substrate rate � possibility to
maintain the entire reactor
length active
� The butane pulses merge
with each other � continuous
butane feed in the 2nd part of
the bioreactor � high rate of
substrate consumption
� Significant substrate
inhibition on contaminant
degradation
↑
↓
convection
dispersion
510=Pe
↓
↑
convection
dispersion
110=Pe
Concentration VS column
heightbutane oxygen
Column height (m)
Co
nc. (m
g L
-1)
0
10
20
30
40
0 0.5 1
butane
oxygen
Column height (m)
Conc. (m
g L
-1)
0
10
20
30
40
0 0.5 1
A crucial aspect for the successful implementation of the pulsed feeding is represented by the
ratio of convection to hydrodynamic dispersion, expressed by the Peclet number:
Longitudinal dispersivity
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT
� To estimate the longitudinal dispersivity of each tested biomass carrier, the 1-L columns were
exposed to tracer tests consisting of pulses of oxygen, TCE or TeCA
0.0
0.2
0.4
0.6
0.8
1.0
0.00 0.02 0.04 0.06 0.08 0.10
Dimensionless time (t/(Vpores/Q))
Dim
ensio
nle
ss
inle
t concentr
ation
Inlet conditions:
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)
� The measured outlet concentrations were interpreted with a 1-D model with advection and dispersion:
Column
1
Column
2
Column
3
Column
4
carrierCerambios Biomax Biomech Biopearl
Effective
porosity0.72 0.64 0.65 0.59
Longitudinal
dispersivity (m)0.050 0.054 0.050 0.040
Exper. Oxygen conc.
Exper. TCE conc.Simulation
Typical results: TCE and oxygen pulsed-
injection with Cerambios as the biofilm carrier
2
2
,z
cD
z
cv
t
c iiL
ii
∂
∂⋅+
∂
∂⋅−=
∂
∂
0.0
0.2
0.4
0.6
0.8
1.0
0 1 2 3 4
0.0
0.2
0.4
0.6
0.8
1.0
0 1 2 3 4
Time / HRT (-)
Dim
ensio
nle
ss o
utlet
conc.
(-)
Dim
ensio
nle
ss o
utlet
conc. (-
)
Time / HRT (-)
vD LiL ⋅≈ α,
with
αL = longitudinal dispersivity
A = section of the column
Φe = effective porosity
OxygenTCE
eA
Qv
φ⋅≈
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)
� With a longitudinal dispersivity of 0.054 m for the selected carrier, a reasonable compromise to avoid an excessive reactor length was found in Pe = 300 total
reactor length = 16 m
� This led to the design of a modular plant, obtained by connecting in series 14 glass columns of 1.2 m of length, each
� The diameter was set to about 0.05 m, to avoid an excessively low value of the ratio of column diameter / carrier diameter
� The reactor total volume resulted therefore equal to 31 L
� To ensure a residence time of 4 days, the groundwater flowrate has to be = 0.2 L/h
� The interstitial velocity results equal to 4 m/d
Reactor design:L
reactorLPe
α≅
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)
Flow sheet of UNIBO’s pilot plant:
1 2 14
Temperature-controlled bath(T = 10-30 °C)
Sampling ports
TCE
saturate
d
solution
Control unit
Oxygen
enrichedwater
Butane enrichedwater
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)
0 300
0,84
1,68
2,52
3,36
4,2
5,04
5,88
6,72
7,56
8,4
9,24
-0,2
0
0,2
0,4
0,6
0,8
1
1,2
time (d)
rTC
E/C
TC
E (
d-1
)
Substrate/Oxygen pulsing schedule applied
TCE conversion attained in the 31-L plant: significantly higher than that attained in the 1-L column; TCE normalized degradation rates (r/C): comparable in the two systems
Goal: optimize the pulsed feeding strategy in order to attain higher TCE biodegradation yields.
SUCCESSFUL SCALE-UP OF THE PROCESS
OPERATING CONDITIONS: - T = 30 °C- HRT = 3.7 - 5.5 days
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)
0 0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
10
5
10
15
20
25
30
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
dimensionless reactor length
Oxygen, Butane [mg/L]
0 0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
10
5
10
15
20
25
30
0
2
4
6
8
10
12
dimensionless reactor length
Oxygen, Butane [mg/L]
THIRD PHASE:• t cycle = 2 days,• Butane pulsed feed = 19% of
the cycle duration, • TCE inlet conc. = 1.37 mg L-1• rTCE/CTCE,in = 0.11 ± 0.02 d-
1• TCE conv. = 49 ± 12%
NINTH PHASE:• t cycle = 4 .66 days, • Butane pulsed feed = 14.3% of
the cycle duration, • TCE inlet conc. = 0.5 mg L-1• rTCE/CTCE,in = 0.19 ± 0.06 d-1• TCE conv. = 76 ± 9%
4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)
CONCLUSIONS
• Preliminary investigation in a 1 L Biomax packed column plant evidenced that the alternated pulse feeding of butane and oxygen leads to an homogenous growth and activity of B4 consortium on Biomax.
Further work is in progress: o to identify biomass carriers characterized by lower longitudinal dispersivities and to
optimize the pulsed substrate/oxygen pulsed feed, in order to attain outlet TCE concentrations as close as possible to the limits for the remediation of CAH-polluted
aquifers;o to test the PBR process at the temperature of the site (15 ° C).
• Butane was selected among 4 tested substrates, and a suspended-cell consortium capable to degrade TCE was developed from the site’s indigenous biomass (B4).
• The attachment of the selected consortium to 4 porous biofilm carriers led to the selection of a ceramic porous carrier (Biomax).
• A 31-L packed-bed reactor, consisting of 14 columns connected in series, was designed, set-up, inoculated and operated for 170 days with satisfactory results; a substrate pulsed feed was set up to avoid excessive inhibition between growth substrate and TCE.
• The high longitudinal dispersivity of the selected carrier determines a significant widening of the butane pulses while they travel along the columns � despite the progressive optimization of the butane/oxygen pulsed feed, substrate inhibition on the TCE biodegradation rate results not negligible.