Spatiotemporal variations of sand hydraulic conductivity by microbial application methods

Viroon Kmhoom ,Thiti Khttiwong ,Treesukon Treeuphtskul ,Surpr Kewswsvong ,Anthony Kwn Leung

a Excellent Centre for Green and Sustainable Infrastructure,Department of Civil Engineering,School of Engineering,King Mongkut’s Institute of Technology Ladkrabang (KMITL),Bangkok,10520,Thailand

b Department of Biomedical Engineering,School of Engineering,King Mongkut’s Institute of Technology Ladkrabang (KMITL),Bangkok,10520,Thailand

c Department of Civil Engineering,Thammasat School of Engineering,Thammasat University,Prathumthani,10200,Thailand

d Department of Civil and Environmental Engineering,The Hong Kong University of Science and Technology (HKUST),Hong Kong,China

Keywords: Bio-mediated soil Dextran Hydraulic conductivity Leuconostoc mesenteroides Microbial application Microstructure

ABSTRACT The spatiotemporal distributions of microbes in soil by different methods could affect the efficacy of the microbes to reduce the soil hydraulic conductivity.In this study,the specimens of bio-mediated sands were prepared using three different methods,i.e.injecting,mixing,and pouring a given microbial solution onto compacted sand specimens.The hydraulic conductivity was measured by constant-head tests,while any soil microstructural changes due to addition of the microbes were observed by scanning electron microscope (SEM) and mercury intrusion porosimetry (MIP) tests.The amount of dextran concentration produced by microbes in each type of specimen was quantified by a refractometer.Results show that dextran production increased exponentially after 5-7 d of microbial settling with the supply of culture medium.The injection and mixing methods resulted in a similar amount and uniform distribution of dextran in the specimens.The pouring method,however,produced a nonuniform distribution,with a higher concentration near the specimen surface.As the supply of culture medium discontinued,the dextran content near the surface produced by the pouring method decreased dramatically due to high competition for nutrients with foreign colonies.Average dextran concentration was negatively and correlated with hydraulic conductivity of bio-mediated soils exponentially,due to the clogging of large soil pores by dextran.The hydraulic conductivity of the injection and mixing cases did not change significantly when the supply of culture medium was absent.

1.Introduction

Contaminant discharge and seepage through reservoir embankments are the most challenging environmental problems in underground construction.Different types of geotechnical structures,especially those constructed on layers of permeable sand,could benefit from methods that can reduce the hydraulic conductivity to mitigate contaminant percolation issues.Conventional ground improvement methods,e.g.chemical grouting,cement stabilisation and pre-treatment of sand before excavation,might contaminate the groundwater and subsurface environment,making them toxic and alkaline (methods using cement).To find more sustainable and environmentally friendly solutions,there has been an increasing interest in the use of bio-materials as soil amenders in recent years,e.g.plants(Umar et al.,2016;Phan et al.,2021;Apriyono et al.,2022;Kamchoom et al.,2022a,2022b),biochar (Chen et al.,2020,2022;Garg et al.,2021) and microbes (Al Qabany et al.,2012;Ayeldeen et al.,2017;Lim et al.,2020).Microbial-induced carbonate precipitation(MICP)has been widely investigated due to its capacity of altering the hydro-mechanical properties (Whiffin et al.,2007;Lin et al.,2016;Konstantinou et al.,2021;Ahenkorah et al.,2023) as well as thermal conductivity (Xiao et al.,2021) of numerous construction materials.In the context of soil improvement,this technique typically involves the injection of ureolytic bacteria,urea,and calcium-rich fluids.The in situ precipitation of calcite between soil particles can significantly enhance the shear strength of soil while decreasing soil hydraulic conductivity (Kantzas et al.,1992;Xu et al.,2023).The MICP has been extensively investigated by various experimental methods,e.g.microfluidic chip test (Chu et al.,2022) and numerical modelling (Wu et al.,2022).However,MICP-treated soil is a brittle material and is susceptible to cracking under certain loading conditions (Wang et al.,2022a),losing the advantage of hydraulic conductivity reduction,especially for events where the soil would experience substantial movements (e.g.underground excavation).

Over the last decade,research has shifted towards more sustainable methods for reducing soil hydraulic conductivity while aiming to have minimal impact on the environment.Microbial cell aggregates,or “biofilm”,are defined as extracellular polymeric substances (EPS).These EPSs promote microbial adhesion to surfaces(e.g.soil grains).The mechanisms of EPS biofilm and MICP are significantly different.According to Harutoshi (2013),EPS is a viscous,slimy polymer that is able to endure ductility strains without fracturing.The EPS biofilm may block the pore media,resulting in “bio-clogging” and a reduction in hydraulic conductivity (Taylor et al.,1990;Rockhold et al.,2002).The bio-clogging process by microbes,includingLeuconostoc mesenteroides(LM),can be effective in both aerobic and anaerobic environments(Zurera-Cosano et al.,2006),and as a result,this method has the potential to treat both shallow and deep soils.

The reduction effect on soil hydraulic conductivity by EPS biofilm varies with the soil environment where the EPS is produced,and also changes with the density of the microbe (Bozyigit et al.,2021).Dennis and Turner (1998) performed a laboratory study to assess the possibility of creating low-permeability barriers using biofilm-treated soil.Even after exposure to a salty or acidic environment,the biofilm’s performance of reducing hydraulic conductivity was observed to be unstable.Chang et al.(2016) used biopolymers that are capable of forming hydrocolloid gel to lower the soil hydraulic conductivity under various confining stresses and pore pressures.The biopolymers in sand can exhibit nearly the same performance under a wide range of confining pressure of 0-400 kPa(i.e.up to approximately 25 m depth in soil).According to Treebupachatsakul and Kamchoom (2021),the bio-clogging process using LM has been demonstrated to be efficient even in the presence of a 50% reduction in nutrients available to the microbes.However,it may not always be the most viable solution as the effectiveness of the method is site-specific.Previous research has focused on carefully prepared specimens of which the preparation method ensures uniform production of EPS across the specimens.In fact,Cunningham et al.(2003)observed a spatial distribution of the microbial cells that were injected into the field.Primarily depending on the method of applying and depositing the microbes in the soil,the spatial distribution of EPS produced could be nonuniform,and the amount of microbial densities could vary(McSwain et al.,2005;Jayathilake et al.,2017).By far,the specimen preparation methods adopted in the literature have no systematic comparison and have a low relevance to engineering applications.To achieve the purpose of hydraulic conductivity reduction in the field,it is important to investigate how microbes might be active and distributed using different application methods that are relevant to engineering practice.

The purpose of this study was to compare the effectiveness of microbes to reduce the hydraulic conductivity of a type of sand using different application methods.Microbial solutions were injected,mixed,and poured on the surface of the soil.The hydraulic conductivity of bio-mediated soil was determined by constanthead tests.The effectiveness of microbes in reducing the hydraulic conductivity was evaluated against other soil amendment methods using cement.At the end of each test,microstructure and pore characteristics were also observed to aid in the data interpretation.

2.Material and methods

2.1.Test plan

A series of laboratory experiments was conducted to determine the temporal variation of soil hydraulic conductivity under the effects of microbes and culture medium.Table 1 summarises all the tests conducted in this study.The first three series of tests aimed to investigate the effects of specimen preparation on any microbialinduced changes in the soil hydraulic conductivity:

(1) The first series(test MI)involved preparing the specimens by injecting 50 mL of microbial solution into the soil;

(2) The specimens in the second series(test MM)were prepared by mixing the microbial solution and culture medium treatment with the soil specimens before compaction,in which the latter one represents the field conditions,as fill compaction is common in building earthen infrastructure;and

(3) The specimens in the third series(test MP)was prepared by pouring 50 mL microbial solution on the surface of compacted soil specimens,which represents the most convenient method in the field without any construction tools.It can also be poured directly on the ground surface before and after construction.

Accounting for natural variability,a total of six replicates were prepared the same way and tested identically in each series.To detect any microbial colonisation,two of these six replicates were used to measure the dextran concentration and observe microbial growth.

The fourth series tested cement-treated soil (test CM),and the results served as a reference for the previous three series.In this series,10% by weight of dry powder of Portland cement was added to the soil prior to compaction.The chosen cement content is representative of that generally used in practice,e.g.grouting and trenching (i.e.5%-10%;Kenney et al.,1992;ACI Committee 230,2009).Given that the culture medium contains water-soluble sucrose,the liquid was more viscous (than water) and could potentially lead to a temporary reduction in soil hydraulic conductivity.To isolate any such effect of culture medium on the soil hydraulic conductivity,the fifth series (test S) tested soil with only the addition of culture medium without microbes.Given that the specimens used for tests CM and S were prepared under wellcontrolled conditions,any major variability of the test results(compared to the cases with the presence of microbes in the previous three series) is not expected.Therefore,no replicate was prepared for these two series.

2.2.Microbial treatment

The microbe used was the commercially available LM TISTR 473 which is a gram-positive strain from Thailand.This genus is abundant in plants and has been used in clinical sources and the food industry.The LM TISTR 473 was classified as risk group 2 by NSTDA (2015),similar to Bacillus sphaericus and Bacillus subtilis that have been used for MICP.Under an excess of carbohydrates,the microbes create environmentally friendly exopolysaccharides,i.e.dextran,as a bio-clogging agent (Wingender et al.,1999).Consequently,such a food-processing by-product(e.g.molasses)likewise may be used in the manufacture of dextran.Depending on their types,microbes may endure temperatures of 10 C-37 C(Hamasaki et al.,2003;Mataragas et al.,2003),meaning that it is an alternative to soil amendment in tropical regions.The sucrose-rich medium was chosen to promote microbial growth (Soetaert et al.,1995; Surasani et al.,2013).As amino acid and vitamin sources,tryptone and yeast extract were used to promote the development of this microbe.LM was transferred from a solid medium(see Fig.1a),and aerobically cultured in a liquid medium containing 10% sucrose,1% tryptone,and 0.5% yeast extract to create the microbial solution.Fig.1b shows that the liquid medium is held in Erlenmeyer flasks and vigorously agitates at a room temperature of about 30 C(Hamasaki et al.,2003)and 150 r/min in an incubator to attain the highest possible growth rate.

Fig.1.Photos of microbial cultivation in(a)solid medium;(b)liquid medium;and(c)a typical growth curve of LM.

The growth curve of LM was determined by the turbidimetric analysis (Dalgaard et al.,1994) at the room temperature of 30 C.Microbes were collected at an interval of every four hours.A Spectrophotometer was used to measure the light absorbance rate(ABS)of the liquid medium and their changes with time.Given that microbial cells impede light from passing through a sample,a greater quantity of microbes means a higher ABS.Fig.1c shows the growth curves of the LM TISTR473.In this study,the optical density(OD) was used to assess the microbial culture that absorbs light with a wavelength of 600 nm(i.e.OD600).The outcome revealed a typical pattern of growth curves.The growth rate of LM TISTR473 experienced a lag during the initial 4 h,indicating that the newly cultivated microorganisms required some time to adjust to the medium (Rofle et al.,2012).Eventually,the growth entered the exponential phase when the cells began to proliferate and divide.This phase is dependent on the metabolic processes that are affected by the temperature and characteristics of the medium(Mataragas et al.,2003).The OD600 of about 1.13 was achieved after 24 h of cultivation.The microbial solution was then transported and applied to each soil specimen.

2.3.Test setup,soil type and specimen preparation

Fig.2 depicts a typical test setup.The specimen containers were made of polyvinyl chloride (PVC) with 700-mm height and 150-mm inner diameter (see Fig.2a).A drainage hole (1 cm in diameter) was drilled at the bottom of each container to facilitate the water outflow,and the hole can be sealed by a PVC plug when the container is water-bathed to control the temperature of the specimen.Prior to compaction,a 50-mm layer of graded pea gravel was placed as a filter to minimise the loss of fine particles as they might migrate during seepage upon repeated cycles of the supply of culture medium and multiple hydraulic conductivity tests.The soil used in this study was uniform and clean Ottawa sand(classified as SP;according to ASTM D2487-17,2017).Index properties of this sand are summarised in Table 2.The sand was oven-dried at 80 C for at least 24 h.Fig.2b shows an overview of the two major phases of specimen preparation,i.e.soil compaction and microbial application.For test MI,the sand was mixed with deionised water to a gravimetric water content of 14% and then compacted to a dry density of (1560 10) kg/m3in five layers using the undercompaction method (Ladd,1978).This corresponds to a degree of compaction of (881)%.After compaction,50 mL of microbial solution was then injected from the soil surface to the middle depth of each specimen using a syringe.The middle depth of the specimen was selected as the injection location for the microbial solution to promote a more even distribution of microorganisms throughout the specimen and prevent the microorganisms from being concentrated primarily at the soil surface or at the bottom of the specimen.To ensure that the microbial solution reached the middle location,measures were taken to control the injection rate at 10 mL/min and to direct the injection towards the centre of the specimen.This was achieved by inserting the needle of a syringe from the soil surface to the middle of the container and gradually injecting the microbial solution under controlled conditions.This procedure attempted to evenly distribute the solution throughout the specimen without causing the development of excess pore water pressure or disturbing the soil structure.To replicate a similar soil structure as accurately as possible,the specimens used for test MM had a gravimetric water content of 14%.Prior to compaction,50 mL of microbial solution was mixed with dry sand,and then deionised water was added until the target gravimetric water content was attained.The microbial solution only made up 1.85%(by volume) and thus it would not introduce significant effects on the overall viscosity of the mixture.The specimen was finally compacted to an identical dry density as test MI.For test MP,the specimen preparation was identical to that for test MI.However,instead of injecting the microbial solution,50 mL was poured directly on the soil surface in the centre of the container.During pouring,the drainage hole at the bottom of the container was left open so that all the solution can permeate to the specimen by displacing the air in the soil pores.After pouring,no solution ponded on the soil surface,and there was no liquid percolating out of the container.Thus,all the microbial solution had entered the specimen.

Fig.2.Overview of (a) the test setup,(b) test plan,and (c) procedures of measuring dextran concentration (dimensions in mm).

Table 2Summary of Ottawa sand properties.

For test CM,a cement content of 10% of the total soil weight was applied.This test series used TPI Cement Type 1,which is ordinary Portland cement made in accordance with the Thai Industrial Standards (TIS 15-2562,2019).For each specimen,the oven-dried sand was thoroughly mixed with the dry cement powder at the targeted content.Deionised water was then added to the dry mix to achieve the same water content as the previous test series,and the wet mixture was compacted in the same manner.Test S has the same preparation method as the tests MI and MP,except that no microbial solution was added.Throughout the experiments,all specimens were kept in a water bath at 29 C 3 C before being taken for hydraulic conductivity tests.The chosen temperature falls within the range of soil temperature variation (i.e.27 C-33 C,Tuntiwaranuruk et al.,2018) in tropical countries,e.g.Thailand.

2.4.Observation of microbial colonisation

Fig.2c depicts the experimental flowchart for the assessment of dextran concentration.In order to examine the microbial colonisation,two replicates were selected from each test series,with one replicate utilised for SEM and MIP tests,and the other for the determination of dextran concentration.A small metal tube(5-mm inner diameter,6-mm outer diameter,150-mm length),which is capable of splitting into two halves,was used.To collect each soil sample,the tube was inserted horizontally through the outlets of each specimen container,into the sampling with length of 5-7 mm approximately.As outlined by Clayton(1986),this method adheres to the length-to-diameter ratio of approximately 1 to minimise disturbance to the soil sample.After collection,the samples were left to freeze-dry with liquid nitrogen,preserving the soil microstructure and hence the pore structure (Sasanian and Newson,2013;Oualmakran et al.,2016).It is important to note that this drying process may have a minor effect on gram-positive bacteria,e.g.LM,as Shinohara et al.(2000)reported that approximately 80% of microbes survived this process.To observe the change in soil microstructure,the microbial colony from samples was collected at the top and bottom locations imaged by a SEM.The MIP tests were conducted on samples collected from the middle location to determine the soil pore size distribution (PSD) and any of its changes due to microbial colonisation for the interpretation of change in the soil hydraulic conductivity.A sample that was collected from a specimen with an identical setup,but without the supply of culture medium and microbial cells (referred to as bare soil),was used as a reference for comparison.The MIP tests were performed on small samples with limited volume.While this approach allows for the observation of the local effects of bacterial installation at different locations within the container,it may not capture the characteristics of the entire soil specimen.Subsequently,the dextran concentration was determined from another replicate using the same procedure for sample collection through the outlets.The samples were oven-dried to determine the water content.The dextran generated by microbial colonisation was then isolated using water solution and centrifugation.Dextran concentration is the ratio of the dextran weight to the total weight of the sample,and the dextran weight was determined by a refractometer(PAL-12S;ATAGO Co.,Ltd.) with an accuracy of 0.2 g.

2.5.Test procedures

Two monitoring stages were performed: the culture medium was supplied and it was absent.The hydraulic conductivity of biomediated soils was determined by constant-head tests (ASTM D2434-19,2019) on four replicates for each test series.The fluid used for this test was deionised water.The test was carried out every four days for the entire duration of 28 d.After the test,1000 mL of liquid culture medium was supplied on top of each specimen and allowed to seep through by gravity(while the bottom drainage was left open).The medium was delivered four times every four days for a total of 16 d.After that,all specimens were left without a supply of culture medium for 14 d.These two phases respectively represented early cultivation and simulated a condition when there was a lack of culture medium.The concentration of dextran in the remaining two replicates was measured every two days.In each of these two replicates,the samples were also collected for the SEM or MIP tests at the end of the two monitoring stages.The culture medium was supplied to test S in the same manner and at the same interval as the first three series.However,there was no culture medium supplied to the specimen in test CM.The hydraulic conductivity of the specimens for tests CM and S was measured simultaneously with the bio-mediated specimens.

3.Results and discussion

3.1.Dextran production and concentration

Fig.3 compares the average dextran concentrations at the top,middle,and bottom of the specimens obtained from tests MI,MM,and MP.During the first 5 d of the supply of the culture medium,only a small increase in dextran was observed in all tests,regardless of the microbial application methods.This consistent response indicated that the microbes were not immediately efficient and required several days to adapt to the soil environment,which can be referred to as the microbial settling period(Rofle et al.,2012).In all tests,the dextran concentration was always the lowest at the top of the specimens compared to other depths,even in test MP (see Fig.3c),where the microbial solution was poured on the surface of the soil sample.This was probably because of the gravity drainage of the liquid solution as it seeped to depths due to the initially high hydraulic conductivity of the sand.

Fig.3.Temporal variations of dextran concentration for tests (a) MI,(b) MM,and (c)MP.

After 5 d of medium supply,the dextran concentration increased exponentially in all the tests because of the metabolic activities of microbes (Mataragas et al.,2003).However,beyond the 15th day,the increasing rates in all cases appeared to slow down,and a steady state of dextran concentration was attained.Evidently,the increasing rates of the dextran concentration at three depths were similar between tests MI and MM.The range of the steady-state dextran concentration at the end of the supply of the culture medium varied between 16% and 20%.Expectedly,for test MI,where the microbial solution was injected at the middle of the container,the dextran production at the middle depth(19%)was the highest,whereas the lowest dextran production (16%) was found near the surface.The close ranges of dextran concentration found between the tests MI and MM suggested that,for the case of sand,applying microbes by means of injection could produce dextran with a similar amount and homogeneity to the means of mixing.For the case of test MP,where the microbial solution was poured on the specimen surface,a substantial increase in the dextran concentration (22%) can be found at the top sampling point,albeit the production of dextran at depths was only half (10%-12%).Indeed,Konstantinou et al.(2021) demonstrated that aerobic and anaerobic environments (especially when soil is saturated in the latter case) were both suitable for dextran production.As a result,the environments at the top,middle,and bottom of the container were equally suitable for microbial growth.The amount of dextran is thus related to the concentration of the microbial solution used in each test.For example,due to the pouring method,the microbial solution in test MP was concentrated in shallow regions rather than at depths.Based on the test results,it is evident that the microbial application method plays an important role in the spatial distributions of the microbial solution (and hence dextran),which could consequently affect the soil hydraulic conductivity.

When the supply of culture medium was ceased for approximately two weeks,the dextran concentration measured from tests MI and MM did not display a substantial drop but appeared to fluctuate about the corresponding steady-state values,although it seemed to be a slight drop near the top for the specimen in test MM.On the contrary,despite the significant increase in the dextran concentration in test MP near the specimen surface,it displayed a huge drop to approximately 1%.Nonetheless,the dextran produced at the other two depths was less affected,with the one at middle depth exhibiting a gradual drop in the concentration.

3.2.Microstructure analysis

The SEM images taken from three tests were compared to understand the variations in dextran concentration.Fig.4a and b shows SEM images taken from test MM at the middle of the container after the absence of culture medium for 12 d.The structure of microbial colonisation (i.e.dextran) is visible and existed as an EPS film(see Fig.4a).The EPS film appears to occupy the soil pore space and cover the soil particles.Closer inspection of the EPS(see Fig.4b)shows that the cocci lenticular form of LM can be found,and this shape resembled the observations made by McCleskey et al.(1947) and Dimic (2006).The majority of LM was grown in solitary,isolated cells beneath the EPS film (see Fig.4b).

Fig.4.SEM images of bio-mediated soils taken from test MM at the middle of the container with magnification of (a) × 500,and (b) × 10,000.

Even in the absence of the culture medium,EPS film can be seen in all tests,especially in the top part of the specimen(see Fig.5a-c).However,some white spots,which are supposed to be foreign colonies,are scattered above the film,especially for those samples obtained from the top location of each container.The foreign colonies appeared to be similar to fungal spores (Aspergillus niger,Priyamvada et al.,2017),which requires oxygen during cultivation and thus can be observed at the top of the soil surface.Foreign colonies were more visible in test MP than in tests MI and MM,according to the SEM images.As the microbial solution was poured on the specimen surface,test MP was expected to have more LM culture and hence more dextran production was found near the surface.However,when the culture medium was absent,foreign colonies presented near the surface were forced to seek alternative nutrient sources.The oxygen availability and high levels of dextran production in test MP provided a favourable environment for these foreign colonies to grow,as evidenced by the SEM analysis (see Fig.5c).This also explains the greater reduction in the dextran concentration (see Fig.3c) in test MP during the period of low nutrient supply.

Fig.5.SEM images of bio-mediated soils taken from the top of the container in (a) test MI,×10,000;(b) test MM,× 5000;(c) test MP,×10,000,and from the bottom of the container in (d) test MI,×1000;(e) test MM,×1000,and (f) test MP,×1000.

As depicted in Fig.5d-f,the SEM images reveal that the samples collected from the bottom of the container in tests MI and MM had a greater quantity of EPS film when compared to that in test MP.This finding matches with the dextran measurements presented in Fig.3,which indicates that the amount of dextran in the samples from the two former tests is 17%-20% approximately,while that in the latter is only halved.Notably,the EPS film found in tests MI and MM covered the voids between the sand particles(see Fig.5d and e),whereas in test MP,the EPS was less prevalent and limited to covering only the surface of the sand particles (see Fig.5f).This difference in EPS amount and location may explain the difference in effectiveness of hydraulic conductivity reduction.

The PSD,which relates differential pore volume (DPV) to pore diameter,is given in Fig.6 for the specimens examined in biomediated soils compared to the reference case (i.e.bare soil).From the reference case (see Fig.6a),a distinct peak emerged at a pore diameter of around 7 μm.Adding microbes by means of injection (test MI) caused a significant reduction in the large pore diameter(i.e.greater than 1 μm),accompanying an increase in the dextran content (Fig.3a),which may had clogged large pores.The results obtained from test MM,where the microbial solution was mixed into the soil,showed a similar PSD to that from test MI,characterised by a peak at the same pore size and a comparable pore volume.However,it should be noted that the pores larger than 10 μm in test MI were better filled with dextran compared to those in test MM.This may be because there was more dextran accumulated in the middle of the container in test MI than in test MM (see Fig.3a and b).There was also an increase in the pore volume between the pore diameter of 0.1 μm and 1 μm.This range of pore diameter is close to the average size of microbial colonies(Kim and Fogler,1999;Ma et al.,2021),suggesting the presence of microbial colonisation.The associated reduction of the large pores was responsible for the significant reduction in the hydraulic conductivity (see Fig.7).

Fig.6.PSD of the bio-mediated soils(a)before and(b)after the cease of the supply of the culture medium.

Fig.7.Temporal variations of the hydraulic conductivity of bio-mediated and cemented soils.

For the case of test MP(see Fig.6a),a similar leftward shift of the PSD can be observed with respect to the reference case,though the shift was less prominent than in the case of tests MI and MM.The large pore diameter (i.e.greater than 5 μm) was reduced.Nevertheless,the pore diameter at the peak pore volume still remained greater than that from tests MI and MM.As the MIP test in test MP was conducted at the middle of the container,the increased amount of dextran content at the middle depth (see Fig.3c) was almost half of that from tests MI and MM (Fig.3a and b).This suggests that the amount of dextran at the middle depth in test MP did not effectively reduce the large pores when compared to the other cases.This is consistent with the distinct characteristics of microbial colonisation within the soil pores as observed through SEM tests (Fig.5d-f) previously discussed.As the supply of the culture medium ceases(see Fig.6b),there is a slight increase in the size of pores greater than 5 μm and this coincides with the reduction in dextran(see Fig.3c).The PSD obtained from the tests MI and MM did not display any significant change,and this observation indicated that the dextran might remain in the pores.

3.3.Effects of microbes on the hydraulic conductivity

Fig.7 compares the temporal variation in the hydraulic conductivity obtained from tests MM,S,and CM.Prior to the application of microbes;the average hydraulic conductivity was 1.2 ×10-4m/s.After 5 d of the supply of the culture medium,the hydraulic conductivity of the bio-mediated soils dropped by almost an order of magnitude from the initial value (reaching an average value of 5.4 ×10-5m/s),despite the production of a rather small amount of dextran(see Fig.3b)during the microbial settling period.Thereafter,following the exponential increase in the dextran content in the following 10 d,the rate of drop in the hydraulic conductivity increased,causing a further reduction by another two orders of magnitude (see Fig.7).The observed drop in hydraulic conductivity was consistent with previous studies(e.g.Dashko and Shidlovskaya,2016) which in general showed that the hydraulic conductivity of coarse-grained soils could be reduced by two to four orders of magnitude following microbial application.For test S,when the microbe was absent yet the culture medium was supplied,the variation of the hydraulic conductivity was marginal(by less than 50%)and was much less compared to the case of test MM.Indeed,the slight amount of the drop was mainly associated with the increased fluid viscosity due to the addition of the culture medium (Wang et al.,2022b).The comparison of the hydraulic conductivity between tests MM and S suggests that the much greater drop in hydraulic conductivity observed in the former case is attributed to the production of microbial dextran.

Compared to the bio-mediated soils,the hydraulic conductivity of sand mixed with 10% cement (i.e.test CM) has reduced greatly since the early days of the application of cement.During the first week,the hydraulic conductivity of the cemented sand reduced by three orders of magnitude to 2.7×10-7m/s,at a much quicker rate than that of the microbe in test MM.However,further reductions in the hydraulic conductivity afterwards were marginal.Despite the faster reduction rate,the steady-state values of hydraulic conductivity between tests CM and MM differed by no more than three times.The comparisons suggested that bio-mediated soils displayed a comparable cementing ability,resulting in a significant reduction in hydraulic conductivity after a sufficiently long period of time.The reduction rate of hydraulic conductivity required to achieve this was relatively slow,compared with the conventional use of cement.Caution should be taken when such a bio-mediation method is used in engineering problems that require a prompt(i.e.within a week) reduction in hydraulic conductivity during construction.

3.4.Effects of microbial application methods

Fig.8 compares the temporal variation in the measured hydraulic conductivity from different microbial application methods.All the tests showed a similar and mild drop in the hydraulic conductivity during the microbial settling period in the first 8 d,following the small production of dextran over the specimens(see Fig.3).After more than a week of application,evidently,the reduction rates of the hydraulic conductivity were observed in all tests,and this is in agreement with the significant production of dextran over the specimens during this period.At the end of the supply of the culture medium,the average hydraulic conductivity obtained from tests MM and MI was reduced to 3.9×10-7m/s and 7.2 × 10-7m/s,respectively,which was much lower than the average value measured from test MP (9.0 × 10-6m/s).In other words,the bio-mediated soils prepared by mixing (test MM) and injection(test MI)displayed a more effective hydraulic conductivity reduction than those prepared by pouring(test MP).In the absence of the culture medium,the hydraulic conductivity obtained from tests MI and MM fluctuated.The average hydraulic conductivity observed in test MM was about 4.2×10-7m/s after the absence of the culture medium for 12 d.Test MI exhibited a slight increase in the average hydraulic conductivity compared to test MM,and it was approximately 1.3×10-6m/s.This may be attributed to the modest reduction in dextran (see Fig.3a) at the top of the specimen examined in test MI.In contrast,the average hydraulic conductivity of test MP appears to increase exponentially to about 5.5×10-5m/s(i.e.almost one order of magnitude) after 12 d.This result was consistent with the substantial reduction in dextran (see Fig.3c).

Fig.8.Temporal variations of the hydraulic conductivity of the bio-mediated soils under different microbe application methods.

Given the uniform dry density along the specimen due to the consistent use of compaction efforts,the presence of dextran mainly in the upper soil layer in test MP could restrict water flow and cause a reduction in the hydraulic conductivity during culture medium supply.This is sufficient to cause an overall reduction in the hydraulic conductivity of the entire specimen.It is important to note that the effectiveness of conductivity reduction is dependent on the proportion of soil affected by the dextran.While foreign colonies were presented in all tests (see Fig.5a-c),the dextran accumulated near the soil surface would provide favourable conditions for the growth of the foreign colonies in the absence of the culture medium.Therefore,when the dextran in the upper soil layer was reduced,this resulted in a substantial increase in the overall hydraulic conductivity in test MP compared to the other tests where the distribution of dextran in the soil specimens was more even.

Fig.9 correlates the hydraulic conductivity with the measured dextran at different locations.The fitted equations and the values ofR2for the correlations between hydraulic conductivity and dextran concentration are also summarised in Table 3.In general,strong negative exponential correlations(R2＞0.88)were always found in all cases except for the bottom case of test MP (R2=0.589).This strong correlation means that the higher the dextran content,the lower the soil hydraulic conductivity,which is consistent with the microscopic observation made in the SEM images (see Fig.5).It is evident that in all cases,the gradient of this semi-logarithm plot obtained from test MP was typically lower than that from tests MM and MP,especially for the correlation obtained from the top and middle of the specimen.It means that for a given production of dextran by the microbes,the specimens treated by surface pouring of the microbial solution were less effective in reducing the hydraulic conductivity.This indicated that soil regions with higher microbial accumulation,such as near the top surface in test MP and the middle location in test MI,did produce more dextran.Nevertheless,these application methods resulted in a non-uniform distribution of dextran at the other depths and did not necessarily lead to a reduction of the hydraulic conductivity.Fig.9d used the average amount of dextran observed at the three locations of the container to correlate with the hydraulic conductivity.The findings demonstrated a considerably better correlation (R2＞0.95) for all tests.

Fig.9.Correlations between the hydraulic conductivity and dextran measured at the(a) top,(b) middle,(c) bottom and (d) average from the three locations.Fitted equations and the corresponding R2 value are summarised in Table 3.

Table 3Summary of fitted equations and R2 for the correlations between hydraulic conductivity and dextran concentration.

The experimental findings suggest that the injection and mixing methods of microbial application were both promising to reduce the soil hydraulic conductivity and maintain it at a reduced level for at least two weeks without the supply of the culture medium.They can be tailored for a broader range of applications,such as the mixing of soil in road construction or injecting near existing structures.The pouring method was found to be less effective in reducing soil hydraulic conductivity than the other two methods,despite being more convenient in the field and less expensive(because no specialised construction tools are required).Moreover,the microbial effects appear to be temporary,especially in the absence of the culture medium supply.As such,the pouring method may also be considered a short-term solution for several applications,including pre-treatment before shallow excavation.

4.Concluding remarks

This study quantified and evaluated the efficiency of using different microbial application methods to the temporal variations of saturated hydraulic conductivity of sand with and without supply of culture medium.A series of controlled laboratory constanthead tests was conducted to measure the hydraulic conductivity of bio-mediated soils prepared in three different ways of injection,mixing,and pouring.The soil mixed with the culture medium but without the microbial solution was tested as a control.The presence of dextran produced by microbes was determined by a refractometer,while the soil microstructural change was observed by SEM and MIP tests.

(1) In all cases,the production of dextran increased exponentially after a period of 5-7 d of microbial settling.Evidently,the pouring method of application introduced a non-uniform distribution of dextran across the depth of the specimen,with much higher dextran content found near the surface.

(2) When the supply of the culture medium was absent for two weeks,in most cases,the dextran remained largely unchanged and apparently attained a steady-state value.However,for specimens prepared by surface pouring,the dextran contents dropped remarkably due to competition for nutrients from foreign colonies.

(3) Among the three application methods,the hydraulic conductivity of bio-mediated soils was negatively exponentially correlated with the amount of dextran(R2＞0.95),especially when the dextran content was taken as the average value across the specimens.

(4) As confirmed by PSDs,the reduction in hydraulic conductivity was associated with the reduction in large pores because of pore-clogging effects due to the formation of EPS film and dextran.The pore volume for the specimens prepared by mixing was reduced more than that by pouring,explaining why the reduction rate of the hydraulic conductivity in the latter case was smaller.

(5) Interestingly,an increase in the hydraulic conductivity was observed for the pouring case following the absence of the culture medium,yet the hydraulic conductivity remained basically unchanged for the injection and mixing cases.In the pouring case,a considerable amount of foreign colonies was found in the specimens because more dextran was produced near the surface.As a result,in the presence of these foreign colonies,the dextran was unable to stay in the large pores,resulting in an increase in hydraulic conductivity.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The first author(V.Kamchoom)acknowledges the grant(Grant No.FRB66065/0258-RE-KRIS/FF66/53) from King Mongkut’s Institute of Technology Ladkrabang (KMITL) and National Science,Research and Innovation Fund(NSRF),and the grant under Climate Change and Climate Variability Research in Monsoon Asia(CMON3)from the National Research Council of Thailand (NRCT) (Grant No.N10A650844) and the National Natural Science Foundation of China (NSFC).The fifth author (A.K.Leung) gratefully acknowledges the funding provided by the Hong Kong Research Grants Council (Grant Nos.GRF#16207521).