Materials and Methods
Bacteria and culture
medium
This study used the ureolytic bacterium Sporosarcina pasteurii(ATCC 11859, from the Guangdong culture collection centre of China), was
used. The culture medium
contained yeast extract 20.0 g/L, polypeptone 10.0 g/L,
(NH4)2SO4 4 g/L, NaCl
8.0 g/L, and distilled water.S. pasteurii was grown in the medium. The medium was prepared
with deionized water and was autoclaved at 121 °C for 20 min. The
initial pH of the medium was 7.0 and the urea concentration was 20 g/L.
Media and inoculants were placed on a shaker table at 30 °C for 48 h of
growth.
Previous research indicated that absorbance and urease activity were
often used to represent the growth characteristic and urealysis ability
of bacteria (Sun et al., 2018a; Sun et al., 2018b;
Stocks-Fischer et al., 1999 ). Changes in cell density were assessed by
monitoring the absorbance (optical density (OD)) of the suspension at a
wavelength of 600 nm (OD600 ) (Fredrickson
et al., 2001 ). The urease activity was measured as the rate of
conductivity increase because of the very strong positive linkage
between the increase of conductivity and urea hydrolysis (Chin
and Kroontje, 1962; Omoregie et al., 2017 ). A bacterial suspension with
OD600 of 1.205 and urease activity of 0.712 mM urea
hydrolysed/min was used for all subsequent tests.
Loess
Loess from the Loess hilly area of Ningxia province in China was used
for all tests. The specific gravity of collapsible loess was 2.721. To
obtain the gradation of loess, particle analysis tests were performed.
The particle size distribution curve is shown in Fig. 1 . The
collapsible loess was mainly medium silty soil and coarse silty soil,
with a clay content of about 20%. The plastic limit was 21.48%, the
liquid limit was 31.34%, and the plasticity index was 9.86. The loess
used here contained calcite (CaCO3), dolomite
(CaMg(CO3)2), Sodium chloride (NaCl),
magnesium sulfate (MgSO4), calcium sulfate
(CaSO4), and other ions.
Experimental
setup
The tests to determine loess slope erosion and the application of
treatment solutions were all conducted in a cube-shaped container
(\(0.18\ m\times 0.24\ m\times 0.04\ m\)), which consisted of
polymethyl methacrylate. The slope angle was fixed at 30° and a
collection vessel was used to collect the lost soil after rainfall
simulation. A schematic view of the setup is shown in Fig. 2 .
Over the course of the tests, pictures were taken by a digital camera to
observe the surface erosion pattern of the loess slopes at 1, 2, 3, 5,
7, 10, 15, 20, 30, 40, and 50 min.
All model slopes were prepared
under identical conditions so that the testing results were comparable;
therefore, the boundary effect was ignored in the tests. During rainfall
simulation, the rainfall intensity was 3 mm/min (180 mm/h) and the pH of
the utilized water was about 7.8. During tests, the water was sprayed
uniformly by a sprayer head on the loess-slope surface.
Treating slope with different
methods
Microbially induced carbonate
precipitation
For convenient comparison, the initial dry density of model slopes was
identical at 1.52 g/cm3. Then, the mixed bacteria
solution and cementation solution (0.75 M of
Ca(Ac)2-urea solution) was uniformly sprayed on the
artificial loess-slope surfaces. Several researchers used
CaCl2 for application of MICP (Mahawish et al.,
2018; Sun et al., 2019a ). Calcium carbonate produced from calcium
chloride is mainly calcite, but
might also be aragonite with calcium
acetate, the bonding effect of which is better than that of calcite
(Tittelboom et al., 2010 ). The amount of bacterial solution
used here was less, therefore, calcium acetate was used to increase the
bonding effect. Khan et al., (2015) reported that cell
densities of bacterial suspension extremely affect the production rates
for precipitation. Apart from the OD600 values, urease
activity of bacteria also affects the cementation effect (Sun et
al., 2018b ). Therefore,
bacterial suspensions with similar OD600 and urease
activity were used for treatment.
Six identical model slope samples were prepared and were divided into
three groups (A, B, and C) according to different volumes of the mixed
solution. Each group had two samples (A1, A2; B1, B2, and C1, C2). For
the subsequent application and promotion of practical engineering, the
amount of the utilized mixed solution was expressed as the spraying
amount per unit area; therefore, the amounts of the mixed solution for
A1, B1, and C1 were 2 L/m2, 4 L/m2,
and 6 L/m2, respectively. With regard to the control
sample in each group, the same amount of distilled water was sprayed
instead. Then, the slopes were dried at 25 °C for 6 days before
subsequent rainfall simulation tests.
Microbially induced carbonate precipitation and Polyvinyl
acetate
To further improve loess slope stability, PVAc (CAS: 9003-20-7) emulsion
was added to the cementation solution. PVAc is a synthetic resin,
prepared by the polymerization of vinyl acetate (Gordon et al.,
2019 ). PVAc emulsion is a type of
water-based and environmentally friendly adhesive (Zhang et al.,
2018 ), which does not negatively impact the environment. Consequently,
PVAc was added for better slope erosion mitigation. The addition of PVAc
to cementation solution might affect the bacterial activity or the MICP
process. Consequently, the effect of the addition of PVAc on bacterial
activity and the production rates of calcium carbonate were
investigated. The amounts of PVAc added to the cementation solution were
20 g/L, 40 g/L, and 60 g/L.
According to the calculation method proposed by Whiffin,
(2004) , 6 ml of bacterial suspension was mixed with 54 ml urea solution
(1 mol/L) and the electrical conductivity was measured every 5 min. The
average change in conductivity per minute (\(ms/cm\bullet min\)) was
calculated and can be converted to the amount of urease hydrolysis per
unit time. Eventually, the rate of hydrolysis of urea per minute
(\(m\text{M\ urea\ hydrolysed}\bullet\min^{-1}\)) was obtained by
multiplying by the dilution factor of 10, which represented enzyme
activity. This method was applied to compare the enzyme activity of the
bacterial suspension after addition of PVAc. The total volume of the
solution was 60 mL; therefore, the amounts of PVAc were 1.2 g, 2.4 g,
and 3.6 g.
To investigate the effect of PVAc addition on the production rates of
calcium carbonate, PVAc was added to the cementation solution (a mixture
of 0.75 M of urea and 0.75 M of calcium) at various amounts (0 g/L, 20
g/L, 40 g/L, and 60 g/L). In this study, the precipitated calcium
carbonate was directly evaluated in transparent polypropylene (PP) tubes
at a temperature of 30 °C. 20 mL of bacterial suspension of S.
pasteurii with an OD600 of 1.205 was mixed with 20 mL
of cementation solution. In a sterile environment, 12 groups of samples
were used, which were divided into four groups according to the amount
of PVAc used. Every group consisted of three parallel samples, all with
an original pH of 7.0. The evaluation criterion was the precipitation
rate of calcium carbonate, i.e., the ratio of the actually produced
amount of calcium carbonate to the theoretically calculated total
amount, which was obtained after a 48-h reaction process. The measuring
method of the actually produced amount of calcium carbonate
has been described in Sun et
al. (2018a) .
During this process, dried samples were weighed and washed with 0.1
mol/L of HCl several times until air bubbles no longer appeared. The
lost dry weight caused by acid leaching was evaluated and was assumed to
be the weight of the actually precipitated CaCO3. The
theoretical total mass of CaCO3 was evaluated by\(C\times V\times M\times t\), where C represents the concentration
of calcium ions in the cementation solution in mol/L, V represents the
solution volume in L, M represents the molar mass of
CaCO3 (100.087 g/mol), and t represents the solidifying
time in days.
To compare the curing effect of PVAc addition on loess slopes, various
amounts of PVAc were added to cementation solution. Five samples (P1,
P2, P3, P4, and P5) were prepared. The amount of mixed solution for
samples was 6 L/m2. The sample P3, P4, and P5 were
treated with MICP, and various amounts of PVAc (20 g/L, 40 g/L, and 60
g/L) were added. P2 was used as MICP control sample and was only treated
with MICP. Sample P1 was a blank control sample, which sprayed the same
amount of distilled water instead. Similarly, the slopes were dried at
25 °C for 6 days before subsequent rainfall simulation tests.
Rainfall Simulation
Test
During the rainfall simulation test, the soil that was washed out from
the model loess-slopes was collected by a collection vessel and the
weight of the loess within the collection vessel was measured at 1, 2,
3, 5, 7, 10, 15, 20, 30, 40, and 50 min.
Surface strength
test
Repeated experiments were conducted with the same samples. These samples
had not experienced rainfall erosion test, but
surface strength test. A soil
penetrometer (type: WISO-750-1, Juchuang company, China) was used to
measure the surface strength of samples. This soil penetrometer could
directly obtain the penetrated depth and surface strength of the soil
via insertion of a probe into the
soil (Ulusay and Erguler, 2012; Miao et al., 2019 ). The depth
of measuring points for surface strengths were all about 0.02 m. The
inserting direction was perpendicular to the soil surface. Six measuring
points were randomly chosen to measure surface strengths.