Delft University of Technology

Applying MICP by denitrification in soils
A process analysis
Pham, Vinh; Nakano, Akiko; van der Star, WRL; Heimovaara, Timo; van Paassen, Leon
DOI
10.1680/jenge.15.00078
Publication date2016
Document Version
Final published version
Published in
Environmental Geotechnics

Citation (APA)
Pham, V., Nakano, A., van der Star, WRL., Heimovaara, T., & van Paassen, L. (2016). Applying MICP bydenitrification in soils: A process analysis. Environmental Geotechnics, 5(2), 79-93. Article 15.00078.
https://doi.org/10.1680/jenge.15.00078
Important note
To cite this publication, please use the final published version (if applicable).
Please check the document version above.

Copyright
Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent
of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons.
Takedown policy
Please contact us and provide details if you believe this document breaches copyrights.
We will remove access to the work immediately and investigate your claim.

This work is downloaded from Delft University of Technology.
For technical reasons the number of authors shown on this cover page is limited to a maximum of 10.

Environmental Geotechnics

Environmental Geotechnics
http://dx.doi.org/10.1680/jenge.15.00078
Paper 15.00078
Received 16/10/2015; accepted 05/10/2016
Keywords: calcium carbonate/geochemistry/strength & testing of materials

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara andvan Paassen

ICE Publishing: All rights reserved

Applying MICP bydenitriﬁcation in soils:
a process analysis
Vinh P. Pham MSc

1

3

PhD Student, Department of Geoscience and Engineering, Delft
University of Technology, Delft, the Netherlands (corresponding
author: p.v.pham-1@tudelft.nl)

Researcher, Subsurface and Groundwater Systems, Deltares, Delft,
the Netherlands
4

Akiko Nakano PhD

2

Wouter R. L. van der Star PhD
Timo J. Heimovaara PhD
Professor, Department of Geoscience and Engineering, Delft Universityof Technology, Delft, the Netherlands

Assistant Professor, Faculty of Agriculture, Kyushu University, Fukuoka,
Japan

5

Leon A. van Paassen PhD
Assistant Professor, Department of Geoscience and Engineering, Delft
University of Technology, Delft, the Netherlands

1

2

3

4

5

The process of microbially induced carbonate precipitation (MICP) by denitriﬁcation was investigated in relation to
its potential use as a ground improvement method. Liquid batch experiments indicated that the substrate solution
had an optimum carbon–nitrogen ratio of 1·6 and conﬁrmed that combining nitrate reduction and calcium carbonate
precipitation leads to an efﬁcient conversion, at which the pH is buffered slightly below 7 and the accumulation of
toxic intermediate nitrogen compounds is limited. Sand column experiments conﬁrmed that the volume anddistribution of the gas phase strongly depend on the stress conditions. The produced gas volume is inversely related
to the pore pressure and can be predicted based on a mass balance analysis, assuming conservation of mass andusing theoretical laws of physics. At low pore pressure, the gas formed and accumulated at the top of the column,
whereas calcium carbonate precipitation occurred mostly at the bottom near the substrate inlet; an excess amountof gas was produced, which vented from the sand columns and induced cracks in the sand at low conﬁning
pressures, which negatively affected the sand-stabilising effect of the calcium carbonate minerals.

Introduction
Microbially induced carbonate precipitation (MICP) has attracted
increased interest in recent years for its potential in geotechnicaland environmental applications (DeJong et al., 2013). Most studies
on MICP were based on the hydrolysis of urea. Hydrolysis of urea
was one of the ﬁrst processes associated with MICP in the latenineteenth century (Ehrlich and Newman, 1996). Due to thelimited abundance of urea in nature, MICP by urea is now
considered to be one of the least important sources of naturally
occurring biogenic carbonate. However, it has been demonstrated
at laboratory scale (Chu et al., 2012; DeJong et al., 2006; Harkes
et al., 2010; Montoya et al., 2013; Whifﬁn et al., 2007) and ﬁeld
scale (Burbank et al., 2011; DeJong et al., 2009; van Paassen,
2011; van Paassen et al., 2010a) that MICP by urea hydrolysis cansigniﬁcantly strengthen granular soils and shows a wide range ofpotential applications (Phillips et al., 2013). Commercial
applications of MICP by urea hydrolysis are still limited, partly
due to the costs of cultivation of ureolytic bacteria and the required

removal of ammonium chloride, which is formed as a by-productof the process. MICP by denitriﬁcation has been considered as apotential alternative biomediated ground improvement process, as
it has several advantages over urea hydrolysis (Karatas et al.,2008; Kavazanjian et al., 2015; van Paassen et al., 2010b). Whennitrate (NO3−) is completely reduced to nitrogen gas, no adverse
by-products are formed and removal effort is not required. The
required substrates for denitriﬁcation are sufﬁciently soluble tolimit the number of injections to reach the target amount ofcalcium carbonate (CaCO3). Substrates can even be produced fromwaste streams, and the conversion does not require cultivation ofvery speciﬁc organisms. In fact, indigenous populations ofdenitrifying bacteria can be used (Martin et al., 2013; van der Staret al., 2009; van Paassen, 2009a; van Paassen et al., 2010b).
Denitriﬁcation is one of the main biological processes in the
global nitrogen cycle, in which a genetically diverse group of
microorganisms is able to reduce nitrate through the intermediate

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

1

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

products of nitrite (NO2−), nitric oxide (NO) and nitrous oxide(N2O) to nitrogen (N2) gas (Robertson and Groffman, 2015).
Denitriﬁcation has been intensively studied since the latenineteenth century, both in natural systems and for industrial
applications (Archna et al., 2012; Ferguson, 1994; Knowles,
1982; Kuenen and Robertson, 1988; Payne, 1981; Soares, 2000;
Voorhees, 1902; Wang et al., 1995).

For zero growth conditions, the overall metabolic stoichiometryis equal to catabolic Reaction I. When following this approach,the actual metabolic stoichiometry ranges between Reactions I
and III. In other words, the amount of required acetate at zero
growth is 0·6 mol/mol nitrate, while the acetate-to-nitrate (Ac/N)
ratio at maximum growth is 1·25 (corresponding to a carbon-tonitrogen (C/N) ratio range of 1·2–2·5). Similarly, the amount of
the produced nitrogen gas ranges between 0·4 and 0·5 mol/mol
nitrate and the inorganic carbon (carbonic acid (H2CO3),
bicarbonate (HCO3−), carbon dioxide (CO2)) ranges from 1·25 to
1·45 mol/mol nitrate.

The stoichiometry of the metabolic reactions involved indenitriﬁcation can be calculated using a method suggested by
Heijnen et al. (1992) and Heijnen and Kleerebezem (2010).
Metabolic reactions can be divided into an anabolic reaction, which
describes the production of biomass, and a catabolic reaction, which
generates the energy for the cells to produce new biomass in the
anabolic reaction (Haynie, 2008). The stoichiometry of the anabolicand catabolic redox reactions are determined separately by solving
the mass and electron balances for each reaction. The ratio between
the catabolic and anabolic reactions is determined by solving theenergy balance – that is, the produced energy from the catabolic
reaction is equal to the energy required for biomass production andcell maintenance. The actual ratio depends on the growth rate of the
microorganisms, which can range from maximum growth
conditions, where microorganisms grow exponentially, to zero
growth conditions, where the total amount of microorganisms does
not increase, but maintaining the population still requires energy.
The actual growth rate is controlled by the process andenvironmental conditions such as the availability of substrates andnutrients and the presence of inhibiting compounds (the conditionof negative growth rate or cell decay, for example in the case where
there are no substrates available, is not considered here).
Using acetate (C2H3O2−) as the electron and carbon donor andnitrate as the electron acceptor leads to the following reaction
stoichiometry for the catabolic reaction

C2 H3 O2− þ 1 6NO3− þ 0  6Hþ
I.
→ 0  8N2 þ 2HCO3− þ 0  8H2 O
Taking CH1·8O0·5N0·2 as a representative molecular formula forbacterial biomass, the anabolic reaction equalsII.

0  725C2 H3 O2− þ 0  2NO3− þ 0  475Hþ
→ CH18 O05 N02 þ 0  45HCO3− þ 0  2H2 O

Solving the energy balance for maximum growth, the stoichiometryof the metabolic reaction (pH 7, temperature 298 K) becomes1 21C2 H3 O2− þ 0  97NO3− þ 0  76Hþ
→ CH18 O05 N02 þ 1  41HCO3− þ 0  39N2 þ 0  59H2 OIII.

2If calcium ions (Ca2+) are present, the inorganic carbon produced
during the metabolism of the denitriﬁers can precipitate to form
calcium carbonate

IV.

Ca2þ þ HCO3− → CaCO3 þ Hþ

At neutral pH, precipitation of calcium carbonate produces
acid, which buffers the alkalinity production associated with
denitriﬁcation. The complete set of coupled reactions related to
MICP by denitriﬁcation, together with the potential consequences
for the physical behaviour of granular soils, is illustrated in
Figure 1. MICP by denitriﬁcation also results in gas production
and biomass growth. Bioﬁlms can also increase the strength andstiffness of soils and reduce hydraulic conductivity (e.g. Stal,2010). Biogenic gas production in soil displaces water, which
may decrease the hydraulic conductivity. The presence of gas in
the pores can also increase shear strength by generating capillarysuction forces (Fredlund and Rahardjo, 1993) and increase theresistance of the soil to cyclic loading (He et al., 2013;
Kavazanjian et al., 2015; Yegian et al., 2007). Unsaturated
conditions may also improve the strengthening effect of MICP, as
the precipitation is considered to take place more efﬁciently
at the particle contacts (Cheng and Cord-Ruwisch, 2012;
Cheng et al., 2013).
Consequently, MICP by denitriﬁcation is considered to havesigniﬁcant potential as a ground improvement method, throughbiogenic gas production and MICP (Hamdan et al., 2016;
Kavazanjian et al., 2015). However, the demonstrated reaction rateof this process is low compared with that of urea hydrolysis.
Selecting the right substrate composition is essential. Too much
nitrate may lead to accumulation of intermediate compounds, which
can be toxic for the bacteria and inhibit growth (Almeida et al.,1995), or increased emissions of nitrous oxide, which is a verystrong greenhouse gas (GHG) (Chung and Chung, 2000; Hanaki etal., 1992), while leaving a large excess of acetate would be
inefﬁcient. Finally, the formation, distribution and mobility of gas
in porous media strongly depend on the soil characteristics andenvironmental conditions. Pore pressure underground strongly
affects the produced gas volume and solubility (e.g. Rebata-Landa
and Santamarina, 2011), and the accumulation and storage of gas is
affected by the substrate supply regime (Soares et al., 1991). If too

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

Representative elementary volume
Gas phase

N2

CO 2
CO 2

Liquid phase

Acid–baseequilibrium

H+

Reduces permeability
and water saturation,
increases the porecompressibility

aNO3– + bC2H3O2– + cH + → biomass + dN2 + eHCO3– + fH2O
Denitrification
H+
CO32– + Ca2+ ↔

Soil particles
Solid phase

Biofilm

Clogging, contributes to cohesion

Precipitation

CaCO3

CaCO3

Increases cohesion and friction, reduces pore size

Figure 1. MICP induced by denitriﬁcation and potential effects in a
representative elementary volume of soil or a soil specimen. Detailed
stoichiometry of the denitriﬁcation reaction is given in the text

much gas is produced, it could trigger liquefaction on very loose
sand instead of mitigating it (Grozic et al., 1998).
For these reasons, this paper presents the results of the following
three sets of experiments that were executed to evaluate theprocess performance of MICP through denitriﬁcation for aselection of process variables and test conditions related to its
application as a ground improvement method.
(a) Batch experiments in a mixed liquid culture in which
substrate composition was varied to study the preferable
substrate ratio and understand the process mechanisms.
(b) Sand column experiments in a triaxial cell, operated at
different pore and conﬁning pressure conditions, to study thegas formation in different pressure conditions correspondent
with different water levels and soil depths.
(c) Sand column experiments at ambient pressure and low
conﬁnement with multiple sequential batches of substrates toinvestigate the precipitation efﬁciency of the treatment.

Materials and methods
Substrate solutions
The substrate solutions used throughout the experiments contained
calcium acetate (Ca(C2H3O2)2) and calcium nitrate (Ca(NO3)2)(Sigma-Aldrich) at varying concentrations (Figure 2). Besides thosemain substrates, all media further contained the following nutrients:
0·003 mM ammonium sulfate ((NH4)2SO4), 0·0024 mM magnesium
sulfate (MgSO4), 0·006 mM monopotassium phosphate (KH2PO4),0·014 mM dipotassium phosphate (K2HPO4) and 1 ml/l trace element
solution SL12B (Overmann et al., 1992) to avoid nutrient limitation
during bacterial growth.

Liquid batch experiments
As an inoculum for the ﬁrst batch experiment, a soil sample
was taken from the Botanical Garden of the Delft Universityof Technology. Denitrifying organisms typically occur in soils
that are wet and anaerobic and have sufﬁciently high organic
content and a source of nitrate, such as wetlands or agriculturalﬁelds (Keddy, 2000). The sample was taken from a depth of 2 musing a hand auger. The black colour and wet condition of
the sample indicated that favourable conditions for denitriﬁers
were met.
The experiments were performed in 250-ml glass bottles (DuranGLS80). Electrodes were inserted through the cap to measure the
pH and electrical conductivity (CE), and tubes were connected tothe sample liquid to monitor the gas volume. In the ﬁrst
incubation, 30 g of soil was suspended in 250 ml of growth
medium. After 3 d of incubation, all nitrate and nitrite wereconsumed and the bottle was shaken to stimulate the detachmentof microorganisms from the soil particles. Afterwards, the liquidcontaining free denitrifying cells (without the solid fraction,
which had been allowed to settle) was collected and transferred
into a new bottle for further experiments. All ﬁve subsequent
incubations used the inoculum from the previous incubation withthe concentrations shown in Figure 2. The substrates used in thesebatches were 30 mM calcium acetate and 25 mM calcium nitrate,
corresponding to calcium, acetate and nitrate (Ca–Ac–N)
concentrations of 55, 60 and 50 mM respectively (55:60:50). In
the ﬁfth batch, calcium salts in one of the two incubations were
replaced by sodium salts (110:60:50 sodium–acetate–nitrate(Na–Ac–N)). In the sixth batch, the Ac/N ratio was varied.
Explicit concentrations of substrates and inoculums of theseexperiments are presented in Figure 2.

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

3

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

Ca–Ac–N 55:60:50:
Ca(C2H3O2)2 30 mM – Ca(NO3)2 25 mM [Ac/N = 1·2]
Ca(C2H3O2)2 20 mM – Ca(NO3)2 25 mM [Ac/N = 0·8]
Ca–Ac–N 45:40:50:
Ca–Ac–N 40:30:50:
Ca(C2H3O2)2 15 mM – Ca(NO3)2 25 mM [Ac/N = 0·6]
Ca–Ac–N 110:120:100: Ca(C2H3O2)2 60 mM – Ca(NO3)2 50 mM [Ac/N = 1·2]
Na–Ac–N 110:60:50:
NaC2H3O2 60 mM – NaNO3 50 mM[Ac/N = 1·2]
All the suspensions contained the same concentrations of micronutrients andtrace elements

First incubation:
30 g soil in 250 ml
liquid.
Ca–Ac–N 55:60:50

Second to fourthincubations: Previous
suspension 0·5 l/l,
Ca–Ac–N 55:60:50

Sixth incubation:
Previous suspension
0·25 l/l,
Ca–Ac–N 40:30:50

Fifth incubation:
Previous suspension
0·25 l/l,
Na–Ac–N 110:60:50

Sixth incubation:
Previous suspension
0·25 l/l,
Ca–Ac–N 45:40:50

Fifth incubation:
Previous suspension
0·25 l/l,
Ca–Ac–N 55:60:50

Sixth incubation:
Previous suspension
0·25 l/l,
Ca–Ac–N 55:60:50

Experiments in the liquid environment
Experiments in the sand columns

Incubation of activatedsludge,Ca–Ac–N 110:120:100

Sand column experiment at ambientpressure, using growth medium
First to third batch Ca–Ac–N 130:160:100
Fourth to ninth batch Ca–Ac–N 120:130:110

Triaxial tests,
Previous suspension
0·25l/l,
Ca–Ac–N 55:60:50
With pressure control

At the ambient pressure

Figure 2. Schematic overview of the sequence of liquid batch
experiments, the ambient pressure column experiment and triaxial
cell experiments. Arrows illustrate the sequence of incubations
and indicate the source of the inoculum for the test

Liquid from the bottles was regularly sampled to analyse theconcentration of different solutes, the electrical conductivity (CE)and the pH. All liquid samples were ﬁltered through a 0·45-µm
membrane and subsequently diluted to the measureable
range before measurements. Nitrate, nitrite and calcium were
determined spectrophotometrically (Lasa 100, Hach Lange) withstandard test kits LCK339, LCK341 and LCK327, respectively).
Total carbon (CT) was measured with a Dohrmann DC190
chromatograph. Acetate consumption was determined indirectlywith total organic carbon (TOC), which is the result from the CT
measurements but pre-treated with hydrochloric acid (HCl) to
remove the inorganic carbon (Schumacher, 2002). All incubations
were performed in a climate room at 25°C. The pH and CE were
measured on all samples and continuously in the incubatingbottles.
The gas produced during the experiment was captured using awater clock, made by placing a graduated cylinder upside down ina water bath. Before starting the incubation, nitrogen gas was
ﬂushed through the bottle to ensure anoxic conditions and thepartial pressure of nitrogen in the gas phase was set at 100%. The
pressure of the gas was kept constant at ambient pressure bymanually adjusting the height of the cylinder. Liquid sampling
and gas volume measurements were done on a daily basis. The
amount of nitrogen gas (NN2) was calculated from the measured
gas volume by using the ideal gas law at 1 atm and 298 K,
assuming nitrogen gas was the only produced gas.
4

Sand column experiments in a triaxial cell at varying
pressure conditions
Sand column experiments were performed at varying pressure
conditions, using a triaxial test set-up described in the ISO/TS
17892-9:2004 standard (CEN, 2004). A uniform ﬁne-grained
siliceous sand (0·125–0·355 mm) was used (Sibelco, S60) with asilica content of 99·5% and a speciﬁc gravity of 2·65 g/cm3. The
minimum and maximum dry bulk densities (ASTM, 2016a,
2016b) ranged from 1·57 ± 0·01 to 1·66 ± 0·02 g/cm3. Sand
columns were prepared using a split mould with a rubber
membrane inside. The mould was ﬁlled in three layers. For eachlayer, ﬁrst, the suspension containing the bacteria and substrates
was poured into the mould, followed by dry sand, ensuring thatthe sand always remained below the ﬂuid level. The sample was
not tamped, thus resulting in a very loosely packed sand with anaverage dry bulk density of 1·50 ± 0·1 g/cm3.
The suspension was prepared by mixing a 0·25 l/l inoculum
harvested at the end of the ﬁfth incubation of the liquid batch
experiments, with concentrated substrate solution and demineralised
water. The overall substrate concentration of the suspension was
30 mM calcium acetate and 25 mM calcium nitrate (Ca–Ac–N
55:60:50). This corresponds to an Ac/N ratio of 1·2, which wasselected to ensure that there would be a sufﬁcient carbon source incase the conditions in the sand would be favourable for bacterial
growth, changing the metabolic stoichiometry towards maximum
growth.

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

The tests were performed at three different pressure conditions.
First, two back pressure conditions were selected to evaluate theeffect of pore pressure on gas volume, while the effective
conﬁning pressure was kept constant: a low back pressure of
50 kPa with a cell pressure of 100 kPa and a high back pressure of
300 kPa with a cell pressure of 350 kPa. Next, a third test was
performed in which the back pressure was set at 345 kPa, while
the cell pressure was kept at 350 kPa to evaluate the process at alow conﬁnement pressure. A control test was performed at a backpressure of 50 kPa and a cell pressure of 100 kPa by using the
growth medium without bacteria.

Consequently, for the other two tests, the volume andconcentrations in the cell water were also determined.

Once the cell and back pressures were set at the designated stresslevels, the sample was left to react while the volume change inthe controllers was monitored. The pore pressure coefﬁcient (or B
factor), which is the ratio of the change in pore pressure over thechange in cell pressure and which is related to the degree of water
saturation of the sample (Skempton, 1954), was measured at the
beginning and at the end of the treatment. At the end of eachexperiment, the sample was ﬂushed from bottom to top withdeaired water by using a third pressure controller, while the backpressure was lowered to obtain a constant head difference of
20 kPa. While ﬂushing, the ﬂow rate was measured to calculate
the hydraulic conductivity of the sample.
After ﬂushing the high-pressure samples, the volume of the
expelled and ﬂushed liquids was determined by emptying theback pressure controller. For the low-pressure conditions, the backpressure controller was emptied before ﬂushing the sample tocollect the ‘expelled’ water and after ﬂushing it was emptied
again to collect the ‘ﬂushed’ water. Samples were taken from theexpelled and ﬂushed liquids to determine the concentrations of
nitrate, nitrite and calcium ions and CT. During the ﬁrst
experiment, biomass growth was observed in the cell water.

Pump

Sand column experiments with multiple batchesof substrate solution
Sand column experiments were performed at ambient pressure
conditions in which multiple batches of substrate solution were
ﬂushed through the columns for the total period of 65 d.
The sand columns were prepared using plastic (polyvinyl
chloride (PVC)) columns with a length of 180 mm and innerdiameter of 65 mm, as described by Harkes et al. (2010).
The column was ﬁlled with a uniform ﬁne siliceous sand (d50:0·166 mm; Itterbeck ﬁne, Smals IKW, SZI 0032). The top andbottom of the column were ﬁlled with 1 cm of ﬁne gravel (d50:
2·5 mm). The sand and gravel were packed by tamping underwater to an average dry density of 1·57 g/cm3. Before treatment,
the sand column was ﬂushed with several pore volumes of
distilled water.
The inoculum for this experiment was obtained from activatedsludge collected at the Harnaschpolder municipal waste water
treatment plant (Delft, the Netherlands). The sludge was suspended
in substrate solution containing 60 mM calcium acetate and 50 mM
calcium nitrate (110:120:100 Ca–Ac–N), and the suspension
was incubated for 6 d, in which nitrate was fully consumed. The
suspension containing the biomass, without the sludge (which
was allowed to settle), was used as the inoculum for the column
set-up. For the column experiment, 250 ml of the suspension
containing the 0·5 l/l inoculum was mixed with substrate solutions,reaching concentrations as indicated in Figure 2, and ﬂushed into
the column. Subsequently, the column was ﬂushed nine times with
250 ml of substrate solution, after which the column was ﬂushedwith one pore volume of distilled water to wash the remaining

Collect every
5–10 minfor analysis

Balance

Flushing under constant headGas and liquid collection between two
flushing times

Figure 3. Set-up of the sand column with multiple substrate
ﬂushes at ambient pressure

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

5

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

Concentration: mMCa-basedNa-based

140

140

120

120

100

100

80

80

60

60

40

40

20

20

0

0

5

10

15

20

25

0

0

CT
[Ca2 + ]5

10

Concentration: mM

(a)
80

80

60

60

40

40

20

20

0

0

5

10

15

20

25

0

0

pH and CE: mS/cm

10

10

8

8

6

6

4

4

2

2
5

20

25
NTotal
NNO3–
NNO2–
NN2

5

(b)

0

15
(d)

10
15
Time: d
(c)

20

25

10

15
(e)

20

25

pH
CE

0

5

10
15
Time: d
(f)

20

25

Figure 4. Comparison of changes in chemical composition during
denitriﬁcation in the presence (left-hand column) and absence(right-hand column) of calcium ions during the ﬁfth incubation.
Legends indicate chemical compounds. (a and d) Results of total

carbon CT and calcium concentration; (b and e) results of nitrate,
nitrite, nitrogen gas and total nitrogen in a volumetric unit; (c andf) results of pH and electrical conductivity CE

solutes from the column. For the ﬁrst three ﬂushes, the Ac/N ratio
was kept high (about 1·6) in order to stimulate bacterial growth andprevent accumulation of toxic intermediate nitrogen compounds.
For all subsequent ﬂushes, the Ac/N ratio was about 1·2, whichcorresponds to the reaction stoichiometry at maximum growth(Reaction III). All liquids were ﬂushed in upward ﬂow under aconstant head difference of 20 cm, and the efﬂuent replaced by
newly ﬂushed medium was collected from the top during each
ﬂush. The set-up is presented in Figure 3.

pH, CE and solute concentrations of these samples were
measured. After each ﬂush, the inlet at the bottom was closed andthe column was left for 7–9 d to react. During this period, theproduced gas and the expelled liquid were collected. The mass ofthe expelled liquid was measured after the reaction period before
the next ﬂush. After treatment, the sand column was ﬁrst analysedusing X-ray computed tomography (CT) scanning, then the PVC
tube was cut open over the length of the sample and removed.
The sample was sliced into nine parts and dried in an oven at
105°C. The dry samples before and after treatment were analysedwith an environmental scanning electron microscope (ESEM)(Philips ESEM XL30). The calcium carbonate concentration wasdetermined for 1·5–2·0 g of the dried samples at different

During each ﬂush, the ﬂow rate was determined by collecting the
efﬂuent from the top of the set-up at regular time intervals andthe hydraulic conductivity was determined using Darcy’s law. The
6

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

locations in the column by using the acid dissolution method
described by Whifﬁn et al. (2007).

excess of calcium and acetate at the end of the experiment. The
Ac/N ratio of 0·6, which corresponds to the stoichiometry of the
catabolic reaction (zero growth; Reaction I), resulted in an excess
of nitrate and accumulation of nitrite. For the Ac/N ratio of 0·8,
all substrates were converted most efﬁciently.

Results
Liquid batch experiments
Figure 4 shows the difference between incubation with calciumbased substrates and sodium-based substrates. The reduction in
CT and calcium ion concentrations in Figure 4(a) and the drop in
CE in Figure 4(c) indicate that calcium carbonate precipitation
took place. Due to the precipitation of carbonate, the pH wasbuffered between 6 and 7, which reduced the accumulation of
nitrite to a maximum level of 10 mM during the ﬁrst week,
compared with 30 mM of nitrite for the sodium-based substratesolution. In the sodium-based system (Figure 4(d)), no
precipitation took place; hence, CT remained constant. The small
amount of calcium ions was due to the small fraction that was stillpresent in the inoculum obtained from the fourth incubation. Due
to MICP, this amount was quickly depleted. The decreases innitrate and nitrite and the increase in nitrogen gas (Figures 4(b)and 4(e)) are evidence that denitriﬁcation still took place in both
systems. In the absence of calcium ions, denitriﬁcation led to an
alkaline environment, as shown in Figure 4(f), where the pH rose
within 3 d to a level between 9 and 9·5.
Figure 5 shows the results of the incubations where the Ac/N ratio
was varied. The experiment using the growth medium with an
Ac/N ratio of 1·2, which is close to the reaction stoichiometry for
maximum growth of the bacteria (Reaction III), resulted in an

Concentration: mM

Ac/N = 1·2

Triaxial tests at different pressure conditions
The volume change in the pressure controllers during the
incubation experiments in the triaxial cell is shown in Figure 6.
The total volume change is the sum of the volume change in thecell and back pressure controllers and represents the change in thesample volume. All tests showed that the volume in the backpressure controller increased rapidly during the ﬁrst 3 d. At thesame time, however, the volume of the cell pressure controller
decreased in all experiments. As a result, the sample volume
decreased before it started to rise. After 4 d, the volume in theback pressure controller of the experiment with a back pressure of
50 kPa continued to rise slowly, while for the two tests withhigher back pressures, the volume started to decrease from thethird day onwards, resulting in a signiﬁcantly lower volume
change at the end of the experiment. The change in sample
volume even became negative at the end of the experiment. The
resulting volume changes and B factors are presented in Table 1.
The volumes and solute concentrations of the different liquid
fractions are presented in Table 2. At low pressure, the backpressure controller indicated that 55 ml was expelled from thesand column. However, the controller contained only 35 ml ofliquid. Similarly, during ﬂushing, 212 ml was ﬂushed out, of
Ac/N = 0·6

Ac/N = 0·8

120

120

120

90

90

90

60

60

60

30

30

30

Concentration: mM

0

0

5

10 15 20 25 30

0

0 5 10 15 20 25 30 35 40

0

TOC[Ca2+]

0 5 10 15 20 25 30 35 40

80

80

80

NTotal
NNO3–

60

60

60

NNO2–
NN2

40

40

40

20

20

20

0

0

5

10 15 20 25 30
Time: d0

0 5 10 15 20 25 30 35 40
Time: dFigure 5. Comparison of changes in chemical composition during
denitriﬁcation in the presence of calcium at three different Ac/N
ratios in the sixth incubation. Left-hand column: Ca–Ac–N =55:60:50, Ac/N = 1·2; middle column: Ca–Ac–N = 45:40:50,

0

0 5 10 15 20 25 30 35 40
Time: dAc/N = 0·8; right-hand column: Ca–Ac–N = 40:30:50, Ac/N = 0·6.
Legends indicate chemical compounds (TOC is total organiccarbon)Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

7

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

to about 3 mS/cm at the bottom, which indicates that, during theﬁnal ﬂushes, a large part of the substrate in the upper half of thecolumn was not yet converted when the next ﬂush was applied.

50–100 kPa
Volume change: ml

60
40
20
0
0

5

10
Time: d(a)
300–350 kPa

15

20

Volume change: ml

10
5
0
–5
–10
0

5

10
Time: d(b)
345–350 kPa

15

20

Total volume
Back volume
Cell volume

Volume change: ml

10
5
0
–5
–10
0

5

10

15

20

During the reaction period between each ﬂush, 31–53 ml of ﬂuid
was expelled from the pores due to gas production inside thecolumn. The hydraulic conductivity, which was calculated from themeasured ﬂow rate during ﬂushing, showed a signiﬁcant difference
between the ﬁrst ﬂush and all other ﬂushes. In the second and later
ﬂushes, initially no liquid ﬂowed out of the column. The injectedliquid ﬁrst ﬁlled up the unsaturated pore space before ﬂowing outof the column. During each ﬂush, the ﬂow rate gradually
increased. The hydraulic conductivity at the end of the second andall later ﬂushes was 50–70% lower that than during the ﬁrst ﬂush.
The increase in hydraulic conductivity can be explained by anincrease in saturation as the trapped gas was gradually ﬂushed out
together with the liquid (increasing the relative permeability).

25

Time: d(c)Figure 6. Volume changes in the triaxial tests at cell pressure –back pressure values of (a) 50–100, (b) 300–350 and(c) 345–350 kPa. The total volume change is the sum of volume
changes recorded by back pressure and cell pressure controllers

which only 207 ml was collected. It was assumed that the rest of
the controller volume was ﬁlled with gas.
Sand column experiment with multiple substrate ﬂushes
at ambient pressure
The breakthrough curves of CE, pH and solute concentrations,which were measured during each ﬂush, are shown in Figure 7.
The initial part of each breakthrough curve can be considered as avertical proﬁle from the top to the bottom through the sandcolumn. The ﬂush was continued until complete breakthrough,indicated by sharp increases in CE and substrate concentrations. In
the ﬁrst two ﬂushes of the fresh medium, the CE proﬁle showed arelatively constant value of 4–5 mS/cm over the height of thecolumn, indicating that there were no differences in soluteconcentration in the whole column. From the fourth ﬂush onwards,
the CE proﬁles started to show a decrease from top to bottom,
reaching maximum values ranging from about 8 mS/cm at the top
8

The solute concentrations show a similar distribution. Nitrate wasalmost completely consumed over the full height of the column inthe ﬁrst four ﬂushes, as only small concentrations of nitrite(<1·3 mM) were measured and about 20 mM calcium was stillpresent. The gradient of the substrate distribution in the column
was very clear in the last two ﬂushes. During these ﬂushes, theremaining nitrate at the top of the column was 20–30 mM(indicating a conversion of only about 50%), while at the bottom it
was completely consumed. The calcium and acetate concentrations
showed a similar distribution. Incomplete nitrate conversion in thelast ﬂushes caused a considerable amount of nitrite accumulation.

The images produced by X-ray CT scanning (Figure 8) show thatthe gas produced during the experiment led to the development of
cracks and open voids, which remained present after the column
was ﬂushed with water. The constructed 3D image in Figure 8(b)shows that the air was mostly present in the pores in the gravel
layers and in the voids, which were developed mostly at the topof the column by the air bubbles themselves. However, as theresolution of the CT scan was limited to approximately 200 mm,
small gas bubbles could not be visualised.
Calcium carbonate measurements showed an average calcium
carbonate content of about 1·1%. However, as shown in Figure 9,
the calcium carbonate was not homogeneously distributed
throughout the column. The distribution of calcium carbonate waslargest at the bottom of the sand layer and decreased towards the
top. Both gravel layers at the top and the bottom and the upper
part of the column had a relatively low calcium carbonate content.
The presence of calcium carbonate was also conﬁrmed by ESEM
analysis (Figure 10). The crystals showed a dendritic texture witha size of up to 200 mm.

Discussion
In order to assess the relevance of these results for ground
improvement applications, ﬁrst, the accuracy of the experiments is
discussed by analysing the mass balance and, second, the effects

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

Experiment

B factor

Controller volume: ml
Day 3Control: 50–100 kPa
300–350 kPa
345–350 kPa
50–100 kPa

Day 15

Back

Cell

Total

Back

Cell

Total

0
6·4
7·4
42·0

−2·2
−3·0
−1·6
−5·5

−2·2
3·4
5·8
36·5

1·3
3·7
1·4
50·0

−4·5
−5·5
−3·5
−7·9

−3·2
−1·8
−2·1
42·1

Hydraulic conductivity:
10−5 m/s

Initial

End

0·98
0·98
0·96
0·58

0·98
0·60
0·86
0·04

5·9
5·9
5·7
5·8

Table 1. Volume changes, B factors and hydraulic conductivities atthe end of the reaction in the triaxial tests

of different process variables and test conditions on the process
performance are evaluated.
Mass balance
Concentration measurements during and after the reaction in each of
the experiments were used to evaluate the mass balance of thedifferent nitrogen compounds. Figures 4 and 5 show the measured
concentrations of the different nitrogen compounds. The nitrogengas concentration was calculated using the ideal gas law assuming
ambient pressure (1 atm) and temperature (298 K) and assuming that
nitrogen gas was the only gas produced and all inorganic carbon
either precipitated or remained in solution due to the relatively highsolubility at neutral to high pH. The gas constant and Henry’s
constant of N2 gas were taken from Kauzmann (2013) and Yaws
(2012). Following these assumptions, the sum of all nitrogencompounds (Ntotal) should be constant and equal to the initial nitrateconcentration. Nevertheless, during the liquid batch experiments, thesum of nitrogen compounds ranged between 40 and 70 mM,

whereas the initial nitrate concentration was 55 mM in each of theexperiments, which corresponds to a deﬁciency in the mass balanceof ±25%.
For the triaxial tests, the total amounts of nitrate and nitrite were
calculated by multiplying the measured concentrations in each of
the liquid fractions by the liquid volume. The total amount of
nitrogen gas was calculated using the ideal gas law, assuming thatthe volume of nitrogen gas was equal to the maximum volume
change in the back pressure controller and that the partial pressureof nitrogen gas was equal to the back pressure. Assuming that thegas phase was in equilibrium with the dissolved nitrogen gas, itsconcentration was calculated using Henry’s law. The resulting
nitrogen balance in the triaxial test is shown in Table 3.
The mass balance calculations in Table 3 show that for the two
experiments under high-pressure conditions, 26 and 33% of thenitrogen compounds in the nitrate added were unaccounted for andExperimentLiquidLiquid volume: ml

Nitrate: mM

Nitrite: mM

Calcium ions: mM

CT: mM

300–350 kPa

Initial suspension
Expelled liquid
Flushed liquid
Cell water
Initial suspension
Expelled liquid
Flushed liquid
Cell water
Initial suspension
Expelled liquid
Flushed liquid
Cell water

97
ND
206
ND
97
ND
206
3630
97
35 (55)a207 (212)a
3633

50·0
ND
2·9
ND
52·7
ND
0
0·15
51·8
1·44
0·11
0·1

0
ND
0·04
ND
0
ND
0
0·15
0
0·01
0
0·158

51·9
ND
0·69
ND
61·6
ND
1·5
0·59
57·6
3·42
1·83
0·66

103·7
ND
9·25
ND
104
ND
7·7
1·73
126
2·2
4·08
0·92

345–350 kPa

50–100 kPaa
The numbers in parentheses are the values recorded in the back pressure controller
ND, not determined

Table 2. Volumes and solute concentrations of different liquids inthe triaxial tests

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

9

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

therefore lost, while for the experiment at (near) ambient pressure,the ﬁnal nitrogen level was 25% higher than initially added. Further
investigation in required to validate the assumptions and explain theobserved discrepancies in the mass balance.

CE: mS/cm

20
15
10
5
0
0

1

2

3

4

5

6

7

8

9

10

5

6

7

8

9

10

5
(c)

6

7

8

9

10

5

6

7

8

9

10

pH

(a)
7·0
6·5
6·0
5·5
5·0
4·5
4·0
0

1

2

3

4

NO2– concentration:mM

NO3– concentration:
mM

(b)
120
100
80
60
40
20
0
0

1

2

3

4

0

1

2

3

4

20
15
10
5
0

Hydraulicconductivity:
cm/s

Ca2+
concentration: mM

(d)
150
100
50
0
0

1

0

1

2

3

4

5
(e)

6

7

8

9

10

2
3
4
5
6
7
8
9
Pore volume (1 pore volume = 230 ml)

10

0·020
0·015
0·010

Also, the C/N ratio in the growth medium signiﬁcantly affected
the conversion efﬁciency. As similarly observed by Chung andChung (2000), this study found that high C/N ratios favoured thecomplete conversion of nitrate and limited nitrite accumulation but
led to an excess of acetate and calcium at the end of theexperiment. A low C/N ratio, on the other hand, led to full acetate
consumption, but at the end of the experiment, signiﬁcantconcentrations of residual nitrate and accumulated nitrite remained.
In this study’s experiments, the optimum Ac/N ratio appears to bearound 0·8, which is slightly lower than the optimum ratio foundby Chung and Chung (2000), but is signiﬁcantly lower than theratio at maximum growth according to Reaction III.

0·005
0

(f)

Figure 7. Measured values of (a) CE, (b) pH, (c) nitrateconcentration, (d) nitrite concentration, (e) calcium ion
concentration and (f) hydraulic conductivity for the sand column
experiment at ambient pressure

10

The effect of substrate composition on process efﬁciency
The results of the liquid batch experiments show that the substratecomposition signiﬁcantly affected the process performance. Whendenitriﬁcation was combined with MICP, the pH was buffered atvalues that are slightly lower than the optimal range for
denitriﬁcation reported in literature (Knowles, 1982; Wang et al.,1995). A similar pH buffering effect by MICP was observed by
Burbank et al. (2011). In their ﬁeld experiments, in which theystimulated MICP by a combination of urea hydrolysis anddenitriﬁcation, Burbank et al. observed that the pH remained
around neutral. The results of this study are partly in agreementwith the observations of Glass and Silverstein (1998), showingthat a high pH – which occurs when sodium-based media are used
– favours the reduction of nitrate to nitrite but can cause nitrite toaccumulate. On the other hand, the solutions containing calcium
showed complete reduction of nitrate, with limited accumulationof nitrite, even though the pH was buffered at a value below 7.
This contradicts the observations of Glass and Silverstein (1998),
who found that nitrate reduction at pH 7 and below wascompletely inhibited. Still, the observed pH values are within therange at which active denitriﬁcation has been reported by others
and far from the lower limit that could cause nitrous oxide
accumulation (Knowles, 1982). Hence, the combined process ofnitrate reduction and calcium carbonate precipitation promotes
efﬁcient conversion of nitrate to nitrogen gas and prevents theaccumulation of toxic intermediates and potential GHGs.

The effect of pressure conditions on gas formation anddistribution
When stimulating MICP by denitriﬁcation in sand, the pressure
conditions signiﬁcantly affected the formation and distribution ofgas in the pore space. For a given amount of substrate, thevolume of gas was much lower at high pore pressures than at low
pore pressures. This is expected, considering that, according to
the ideal gas law, the volume and pressure of gas are inversely
proportional. Secondly, according to Henry’s law, gas solubility ishigher at high pressures. Thirdly, a higher gas solubility leads to

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

higher dissolved gas concentrations, which may lead to larger
diffusive ﬂuxes to surrounding water, as observed in the case of
300–350 and 345–350 kPa in Figure 6.

(shallow depth), the gas may lead to the formation of cracks, disturb
the sand structure and reduce the stabilising effect of the calcium
carbonate minerals, as shown in Figure 8.

The total gas volume in the pore space signiﬁcantly inﬂuences thegeotechnical properties of sand, including strength, permeability andstiffness (DeJong et al., 2013). After a speciﬁc amount of gas is
produced, single gas bubbles will coagulate into gas pockets or form
a continuous gas phase, which tends to migrate upwards in irregularpatterns (Haines, 1930; van Paassen, 2010b). Also, the conﬁnement
pressure inﬂuences the potential of MICP by denitriﬁcation forground reinforcement applications. At low conﬁnement conditions

The relationship between the distribution of gas andcalcium carbonate
The average calcium carbonate content in the sand column which
was ﬂushed nine times with substrate solution reached 1·1%.
Although this amount of calcium carbonate is relatively low
compared with the results from other studies on MICP based onhydrolysis of urea, such as that of van Paassen et al. (2010a), it
may still be sufﬁcient to signiﬁcantly increase cyclic shear

(a)

(b)

Figure 8. Result of the gas distribution in the sand column
treated at ambient pressure with multiple substrate ﬂushes:(a) X-ray CT scanned images; (b) constructed three-dimensional
image

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

11

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

6·5 cm

0·40%

0·74%

0·89%

18 cm
1·05%

1·02%

1·00%
1·09%
1·53%
0·88%

Figure 9. Calcium carbonate distribution in the sand column
treated at ambient pressure

resistance to mitigate liquefaction of loose, sandy soils (DeJong etal., 2014; Kavazanjian et al., 2015). The relationship between
strength and calcium carbonate content depends on many factors,

200 µm
Acc.V Spot Magn Det WD Exp
16·0 kV 5·0 100x BSE 9·7 1
1·0 Torr sand treatedincluding the density, mineral type and grain size distribution of
the treated sand (Cheng et al., 2013; van Paassen et al., 2010a)
and substrate concentrations, reaction rate and environmental
conditions of the treatment process (Al Qabany et al., 2012;
Mortensen et al., 2011; van Paassen, 2009a).
Gas production can have a positive effect on the strength gain due
to MICP. Cheng and Cord-Ruwisch (2013) showed that columns
that have been treated under partially saturated conditions require
lower amounts of calcium carbonate to obtain a similar increase instrength for fully saturated conditions. They considered that, inpartially saturated conditions, substrate solutions preferably ﬁll thepore throats due to suction effects, resulting in a more efﬁcient
distribution of cementing calcium carbonate minerals. Similarly,
Kavazanjian et al. (2015) showed that MICP by denitriﬁcation
results in more efﬁcient cementation than MICP by urea hydrolysis.
The present study showed that, within the sand column treated atambient pressure, calcium carbonate was predominantly formed at
the bottom of the column, while gas was predominantly formedat the top. This seems obvious because there is less substrateavailable to react when gas displaces the substrate solution. As aresult, the potential improvement in the cementation efﬁciency due
to desaturation is counteracted by a lower conversion efﬁciency.
Secondly, the cracks that were formed at low conﬁning pressure
disturb the sand-stabilising effect of the calcium carbonate minerals.
The low number, large size and dendritic structure of the calcium
carbonate crystals could be due to the fact that the crystals grew in
multiple phases at a relatively low reaction rate (van Paassen,
2009b) and in the presence of impurities such as organic polymers
from the growing biomass.
Conversion rate
The overall reaction rate of denitriﬁcation and precipitation is an
important process parameter, which depends on many factors,
including the amount and type of denitrifying organisms, substrate
and product concentrations and environmental effects, such as

Acc.V Spot Magn Det WD Exp
16·0 kV 5·0 100x BSE 9·9 1

Figure 10. Presence of calcium carbonate in the sand treated withmultiple substrate ﬂushes at ambient pressure in ESEM images

12

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

20 µm
1·0 Torr sand treatedEnvironmental Geotechnics

Initial: mmol nitrogen
Final: mmol nitrogen

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

300–350 kPa

345–350 kPa

50–100 kPa

4·85
ND
ND
0·6
0·01
2·14
0·51
3·26
1·59
33

5·11
0·55
0·54
0
0
2·13
0·57
3·78
1·33
26

5·03
0·36
0·57
0·1
0
5·18
0·19
6·40
−1·37
−25

Nitrate in the suspension
Nitrate in cell water
Nitrite in cell water
Nitrate in (ﬂushed + expelled) water
Nitrite in (ﬂushed + expelled) water
Nitrogen gas
Nitrogen gas, aqueous
Total

Ngap: mmol
Ngap: %
ND, not determined

Table 3. Nitrogen balance for triaxial tests

temperature, pH, salinity and the presence of other catalysing or
inhibiting compounds (van Paassen, 2009a). Reported reaction rates
range from a few millimoles of nitrate per litre per day up to
170 mmol nitrate/(l d) (Glass and Silverstein, 1998; Martin et al.,2013; Soares et al., 1991; van Paassen et al., 2010b). This study
showed that the reaction rate in the triaxial cell was about
10–15 mmol nitrate/(l d). The batch experiments in liquid
environment in which the same substrate concentrations andinoculum size were used showed a rate of 1–2 mmol nitrate/(l d),about ﬁve to eight times lower than those for the triaxial cell. The
average reaction rate in the sand column at ambient pressure wasabout 6 mmol nitrate/(l d). However, the remaining substrates in theﬁnal ﬂushes indicated that the reaction rate in the unsaturated top
part of the column was lower than that in the saturated bottom part.
Considering that at least a small mass percentage of calcium
carbonate needs to be formed, it is expected that, based on thecurrent results, several weeks to several months of treatment are
still required in order to obtain sufﬁcient strength improvement
when using MICP through denitriﬁcation. Further investigation isrequired to explain the observed differences in reaction rate.

Conclusions
This study has shown that the combined process of biologicaldenitriﬁcation and MICP leads to a more efﬁcient conversion thandenitriﬁcation without precipitation. The precipitation of calcium
carbonate buffers the pH and prevents the accumulation of toxic
intermediates. The optimum acetate-to-nitrate (Ac/N) ratio, at
which all substrates are consumed most efﬁciently, is about 0·8(which corresponds to a carbon-to-nitrogen ratio of 1·6). Lower
Ac/N ratios lead to the accumulation of toxic intermediate nitrogencompounds, while higher ratios leave signiﬁcant amounts ofresidual calcium and acetate. Sand column experiments conﬁrmedthat the volume and distribution of the gas phase strongly dependon the pressure conditions. The produced gas volume is inversely
related to the pore pressure and can be reasonably predicted using
a generic thermodynamic approach, which solves the mass andcharge balance to determine the stoichiometry of the metabolic

reaction, and making some simplifying assumptions to determine
the distribution of gas over the dissolved and gas phases. Further
investigation is required to explain the observed discrepancy
between measured and expected values. Under high-pore pressure
conditions in the triaxial cell, the produced gas initially expelled
the liquid phase but later on seemed to dissipate from the sandsample, probably by diffusion through the latex membrane. At low
pore pressures, the produced gas was higher than the gas storage
capacity of the sand column and escaped to the back pressure
controller column while expelling part of the liquid phase. In thesand column treated with multiple batches of substrate solution atambient pressure, the conversion rate and cumulative amount ofcalcium carbonate appeared to be inversely related to the gas
distribution because low conﬁnement cracks were induced, which
may negatively inﬂuence the sand-stabilising effect of the calcium
carbonate minerals.

Acknowledgements
This study was supported by the International Training Programand Institutional Program of Young Researcher Overseas Visits
launched by the Japan Society for the Promotion of Science(JSPS) and the Dutch Technology Foundation STW, which is partof the Netherlands Organisation for Scientiﬁc Research, and waspartly funded by the Dutch Ministry of Economic Affairs, through
STW perspective program BioGeoCivil (11337) and JSPS Grantin-Aid for Young Scientists (B) Grant Number 25850166.
REFERENCES

Al Qabany AA, Soga K and Santamarina C (2012) Factors

affecting efﬁciency of microbially induced calcite
precipitation. Journal of Geotechnical and Geoenvironmental
Engineering 138(8): 992–1001, http://dx.doi.org/10.1061/(ASCE)GT.1943-5606.0000666.
Almeida JS, Julio SM, Reis MAM and Carrondo MJT (1995) Nitrite
inhibition of denitriﬁcation by Pseudomonas ﬂuorescens.
Biotechnology and Bioengineering 46(3): 194–201, http://dx.
doi.org/10.1002/bit.260460303.

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

13

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

Archna, Sharma SK and Sobti RC (2012) Nitrate removal from

Ferguson SJ (1994) Denitriﬁcation and its control. Antonie vanground water: a review. E-Journal of Chemistry 9(4):1667–1675, http://dx.doi.org/10.1155/2012/154616.
ASTM (2016a) D 4253-16: Standard test methods for
maximum index density and unit weight of soils using avibratory table. ASTM International, West Conshohocken,
PA, USA.
ASTM (2016b) D 4254-16: Standard test methods for minimum
index density and unit weight of soils and calculation ofrelative density. ASTM International, West Conshohocken,
PA, USA.

Leeuwenhoek 66(1–3): 89–110, http://dx.doi.org/10.1007/
BF00871634.
Fredlund DG and Rahardjo H (1993) Soil Mechanics for
Unsaturated Soils. Wiley, New York, NY, USA.
Glass C and Silverstein J (1998) Denitriﬁcation kinetics of highnitrate concentration water: pH effect on inhibition and nitrite
accumulation. Water Research 32(3): 831–839, http://dx.doi.
org/10.1016/S0043-1354(97)00260-1.
Grozic JL, Robertson PK and Morgenstern NR (1998) The
behavior of loose gassy sand. Canadian Geotechnical Journal
36(3): 482–492, http://dx.doi.org/10.1139/t99-007.
Haines WB (1930) Studies in the physical properties of soils: V.
the hysteresis effect in capillary properties, and the modes ofwater distribution associated therewith. Journal of Agricultural
Sciences 20(1): 97–116.
Hamdan N, Kavazanjian E, Rittmann BE and Karatas I (2016)
Carbonate mineral precipitation for soil improvement throughmicrobial denitriﬁcation. Geomicrobiology Journal, http://dx.
doi.org/10.1080/01490451.2016.1154117.
Hanaki K, Hong Z and Mastsuo T (1992) Production of nitrous
oxide gas during denitriﬁcation of wastewater. Water Scienceand Technology 26(5–6): 1027–1036.

Burbank MB, Weaver TJ, Green TL, Williams BC and Crawford RL(2011) Precipitation of calcite by indigenous microorganisms
to strengthen liqueﬁable soils. Geomicrobiology Journal 28(4):301–312, http://dx.doi.org/10.1080/01490451.2010.499929.
CEN (Comité Européen de Normalisation) (2004) ISO/TS 178929:2004: Geotechnical investigation and testing – laboratory
testing of soil – part 9: consolidated triaxial compression tests
on water saturated soil. CEN, Brussels, Belgium.
Cheng L and Cord-Ruwisch R (2012) In situ soil cementation withureolytic bacteria by surface percolation. EcologicalEngineering 42: 64–72, http://dx.doi.org/10.1016/j.ecoleng.
2012.01.013.
Cheng L, Cord-Ruwisch R and Shahin MA (2013) Cementation ofsand soil by microbially induced calcite precipitation at
various degrees of saturation. Canadian Geotechnical Journal
50(1): 81–90, http://dx.doi.org/10.1139/cgj-2012-0023.
Chu J, Stabnikov V and Ivanov V (2012) Microbially induced
calcium carbonate precipitation on surface or in the bulk ofsoil. Geomicrobiology Journal 29(6): 544–549, http://dx.doi.
org/10.1080/01490451.2011.592929.
Chung YC and Chung MS (2000) BNP test to evaluate theinﬂuence of C/N ratio on N2O. Water Science and Technology
42(3–4): 23–27.
DeJong JT, Fritzges MB and Nusslein K (2006) Microbiallyinduced cementation to control sand response to undrained
shear. Journal of Geotechnical and Geoenvironmental
Engineering 132(11): 1381–1392, http://dx.doi.org/10.1061/(ASCE)1090-0241(2006)132:11(1381).
DeJong JT, Martinez BC, Mortensen BM et al. (2009) Upscalingof bio-mediated soil improvement. In 17th International
Conference on Soil Mechanics and Geotechnical Engineering(Hamza M, Shahien M and El-Mossallamy Y (eds)). IOS
Press, Amsterdam, the Netherlands, pp. 2300–2303.
DeJong JT, Soga K, Kavazanjian E et al. (2013) Biogeochemical
process and geotechnical applications: progress, opportunities
and challenges. Géotechnique 63(4): 287–301, http://dx.doi.
org/10.1680/geot.SIP13.P.017.
DeJong JT, Martinez BC, Ginn TR et al. (2014) Development of ascaled repeated ﬁve spot treatment model for examining
microbial induced calcite precipitation feasibility in ﬁeld
applications. Geotechnical Testing Journal 37(3): 424–435,
http://dx.doi.org/10.1520/GTJ20130089.
Ehrlich HL and Newman DK (1996) Geomicrobiology, 3rd edn.
CRC, Boca Raton, FL, USA.
14

Harkes MP, van Paassen LA, Booster JL, Whiffen VS and van
Loosdrecht MCM (2010) Fixation and distribution of bacterial

activity in sand to induce carbonate precipitation for groundreinforcement. Ecological Engineering 36(2): 112–117,
http://dx.doi.org/10.1016/j.ecoleng.2009.01.004.
Haynie DT (2008) Biological Thermodynamics. Cambridge
University Press, Cambridge, UK.
He J, Chu J and Ivanov V (2013) Mitigation of liquefaction ofsaturated sand using biogas. Géotechnique 63(4): 267–275,
http://dx.doi.org/10.1680/bcmpge.60531.011.
Heijnen JJ and Kleerebezem R (2010) Bioenergetics of
microbial growth. In Encyclopedia of Industrial
Biotechnology: Bioprocess, Bioseparation and Cell
Technology (Flickinger MC (ed.)). Wiley, New York,NY, USA, vol. 1, pp. 594–617.
Heijnen JJ, van Loosdrecht MCM and Tijhuis L (1992) A black
box mathematical model to calculate auto- and heterotrophic
biomass yields based on Gibbs energy dissipation.
Biotechnology and Bioengineering 40(10): 1139–1154,
http://dx.doi.org/10.1002/bit.260401003.
Karatas I, Kavazanjian E and Rittmann B (2008) Microbiallyinduced precipitation of calcite using Pseudomonas
denitriﬁcans. Proceedings of 1st International Conference on
Bio-geo-civil Engineering, Delft, the Netherlands, pp. 58–66.
Kauzmann W (2013) Kinetic Theory of Gases. Dover,
Newburyport, MA, USA.
Kavazanjian E, O’Donnell ST and Hamdan N (2015)
Biogeotechnical mitigation of earthquake-induced soilliquefaction by denitriﬁcation: a two-stage process.
Proceedings of 6th International Conference on Earthquake
Geotechnical Engineering, Christchurch, New Zealand,
pp. 20–28.

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

Environmental Geotechnics

Applying MICP by denitriﬁcation in soils:
a process analysis
Pham, Nakano, van der Star, Heimovaara and van Paassen

Keddy PA (2000) Wetland Ecology – Principle and Conservation.

van der Star WRL, Taher E, Harkes MP et al. (2009) Use of waste

Cambridge University Press, Cambridge, UK.
Knowles R (1982) Denitriﬁcation. Microbiological Reviews 46(1):43–70.
Kuenen JG and Robertson LA (1988) Ecology of nitriﬁcation anddenitriﬁcation. In SSGM 42: The Nitrogen and Sulphur(Society for General Microbiology Symposia) (Cole JA andFerguson SJ (eds)). Cambridge University Press, Cambridge,
UK, pp. 161–218.
Martin D, Dodds K, Butler IB and Ngwenya BT (2013) Carbonate
precipitation under pressure for bioengineering in theanaerobic subsurface via denitriﬁcation. Environmental
Science and Technology 47(15): 8692–8699, http://dx.doi.org/
10.1021/es401270q.
Montoya BM, Dejong JT and Boulanger RW (2013) Dynamic
response of liqueﬁable sand improved by microbial-induced
calcite precipitation. Géotechnique 63: 267–275.

stream and microbes for in situ transformation of sand into
sandstone. In Ground Improvement Technologies and Case
Histories (Leung CF, Chu J and Shen RF (eds)). Research
Publishing Services, Singapore, pp. 177–182, http://dx.doi.org/
10.3850/GI126.
van Paassen LA (2009a) Biogrout – Ground Improvement by
Microbially Induced Carbonate Precipitation. PhD thesis,
Delft University of Technology, Delft, the Netherlands.
van Paassen LA (2009b) Microbes turning sand into sandstone,
using waste as cement. Proceedings of 4 iYGCE’09 – 4thInternational Young Geotechnical Engineers’ Conference,
Alexandria, Egypt, pp. 135–138.
van Paassen LA (2011) Bio-mediated ground improvement: fromlaboratory experiment to pilot applications. In Geo-frontiersCongress 2011: Advances in Geotechnical Engineering (Han J
and Alzamora DE (eds)). American Society of Civil
Engineers, Reston, VA, USA, pp. 4099–4108.

Mortensen BM, Haber MJ, Dejong JT, Caslake LF and Nelson DC(2011) Effects of environmental factors on microbial induced
calcium carbonate precipitation. Journal of Applied
Microbiology 111(2): 338–349, http://dx.doi.org/10.1111/j.
1365-2672.2011.05065.x.
Overmann J, Fischer U and Pfennig N (1992) A new purple sulfur
bacterium from saline littoral sediments, Thiorhodovibrio
winogradskyi gen. nov. and sp. nov. Archives of Microbiology
157(4): 329–335, http://dx.doi.org/10.1007/BF00248677.
Payne WJ (1981) Denitriﬁcation. Wiley, New York, NY, USA.
Phillips AJ, Gerlach R, Lauchnor E et al. (2013) Engineered
applications of ureolytic biomineralization: a review.
Biofouling 29(6): 715–733, http://dx.doi.org/10.1080/
08927014.2013.796550.
Rebata-Landa V and Santamarina JC (2011) Mechanical effects ofbiogenic nitrogen gas bubbles in soils. Journal of Geotechnicaland Geoenvironmental Engineering 138(2): 128–137,
http://dx.doi.org/10.1061/(ASCE)GT.1943-5606.0000571.
Robertson GP and Groffman PM (2015) Nitrogentransformations. In Soil Microbiology, Ecology andBiochemistry, 4th edn. (Paul EA (ed.)). Academic Press,
Boston, MA, USA, pp. 421–446.
Schumacher BA (2002) Methods for the Determination of Total
Organic Carbon (TOC) in and Sediments. US Environmental
Protection Agency, Washington, DC, USA.
Skempton AW (1954) The pore-pressure coefﬁcients A and B.
Géotechnique 4(4): 143–147, http://dx.doi.org/10.1680/geot.
1954.4.4.143.
Soares MIM (2000) Biological denitriﬁcation of groundwater.
Water, Air, and Soil Pollution 123(1–4): 183–193, http://dx.
doi.org/10.1023/A:1005242600186.
Soares MI, Braester C, Shimshon B and Abeliovich A (1991)
Denitriﬁcation in laboratory sand columns: carbon regime, gas
accumulation and hydraulic properties. Water Research 25(3):325–332, http://dx.doi.org/10.1016/0043-1354(91)90013-G.
Stal LJ (2010) Microphytobenthos as a biogeomorphological force inintertidal sediment stabilization. Ecological Engineering 36(2):236–245, http://dx.doi.org/10.1016/j.ecoleng.2008.12.032.

van Paassen LA, Ghose R, van der Linden TJM, van der Star WRL
and Loosdrecht MCM (2010a) Quantifying biomediatedground improvement by ureolysis: large-scale biogrout
experiment. Journal of Geotechnical and Geoenvironmental
Engineering 136(12): 1721–1728, http://dx.doi.org/10.1061/(ASCE)GT.1943-5606.0000382.
van Paassen LA, Daza CM, Staal M et al. (2010b) Potential soil
reinforcement by biological denitriﬁcation. EcologicalEngineering 36(2): 168–175, http://dx.doi.org/10.1016/j.
ecoleng.2009.03.026.
Voorhees EB (1902) Studies in denitriﬁcation. Journal of theAmerican Chemical Society 24(9): 785–823.
Wang JH, Baltzis BC and Lewandowski GA (1995) Fundamental
denitriﬁcation kinetic studies with Pseudomonas denitriﬁcans.
Biotechnology and Bioengineering 47(1): 26–41, http://dx.doi.
org/10.1002/bit.260470105.
Whifﬁn VS, van Paassen LA and Harkes MP (2007) Microbialcarbonate precipitation as a soil improvement technique.
Geomicrobiology Journal 24(5): 417–423, http://dx.doi.org/10.
1080/01490450701436505.
Yaws CL (2012) Yaws’ Handbook of Properties for Aqueous
Systems. Knovel, New York, NY, USA.
Yegian MK, Eseller-Bayat E and Alshawabkeh A (2007) Inducedpartial saturation for liquefaction mitigation: experimental
investigation. Journal of Geotechnical and Geoenvironmental
Engineering 133(4): 372–380, http://dx.doi.org/10.1061/(ASCE)1090-0241(2007)133:4(372).

HOW CAN YOU CONTRIBUTE?

To discuss this paper, please submit up to 500 words
to the editor at journals@ice.org.uk. Your contribution
will be forwarded to the author(s) for a reply and, if
considered appropriate by the editorial board, it will be
published as a discussion in a future issue of the journal.

Downloaded by [ TU Delft Library] on [03/11/16]. Copyright © ICE Publishing, all rights reserved.

15