Bioclogging in porous media: influence in reduction of hydraulic conductivity and organic contaminants during synthetic leachate permeation
© Kanmani et al.; licensee BioMed Central Ltd. 2014
Received: 30 July 2013
Accepted: 14 October 2014
Published: 29 October 2014
In this study the concept of biofilm accumulation in the sand column was promoted to assess the changes in hydraulic conductivity and concentration of organic contaminants of the synthetic leachate. Four different combinations of column study were carried out using synthetic leachate as a substrate solution. Mixed and stratified mode of experiments with two different sizes (0.3 mm and 0.6 mm) of sand grains were used for column filling. Two columns were acting as a blank, the remaining two columns amended with mixed microbial cultures which were isolated from leachate. The column was operated with continuous synthetic leachate supply for 45 days. The results indicated that the highest hydraulic conductivity reduction occurred in the mixed sand microbial column with 98.8% when compared to stratified sand microbial column. The analysis of organic contaminants of the effluent leachate was also clearly shown that the mixed sand amended with microbes poses a suitable remedial measure when compared to natural and synthetic liners for controlling the leachate migration in the subsurface environment.
KeywordsBiofilm Hydraulic conductivity Microbial culture Synthetic leachate
The generation of solid waste has become an increasing environmental and public health problem everywhere in the universe, especially in the developing countries . Open dumps are the oldest and the most common mode of disposing of solid waste. Although in recent years, thousands have been closed, many still are being used . The dumping of solid waste in uncontrolled landfills can have significant impacts on the environment and human health . Leachate migrations from waste sites or landfills and the release of pollutants from sediment (under certain conditions) pose a high risk to groundwater resource if not adequately managed. Plenty of materials have been identified as contaminants of ground water. These include synthetic organic chemicals, hydrocarbons, inorganic cations, inorganic anions, pathogens, and radio nuclides. Most of these materials dissolve in water to varying degrees . Their impact on groundwater continues to raise concern and have become the subject of recent and past investigations -.
Continuously increasing awareness in preserving the groundwater supplies from contaminants generated from waste sites has given rise to the design of well-isolated containment structures. These measures generally involve the application of low permeability natural clays and sand-bentonite mixtures or synthetic materials . Compacted natural clays are often used in constructing hydraulic barriers underneath waste containment systems. The hydraulic conductivity must be less than or equal to 1 × 10‒7 cm/s for soil liners and covers used to contain hazardous waste, industrial waste, and municipal waste . In the absence of impervious natural clay liners, Geosynthetic Clay Liners (GCLs) are progressively being employed as constituent of a composite liner with geomembranes (GM) in landfill barrier systems . The primary advantages of the GCL are the limited thickness, the good compliance with differential settlements of the underlying ground or waste, easy installation and low price. On the other hand, the limited thickness of this barrier can produce: (1) vulnerability to mechanical accidents, (2) limited sorption capacity, and (3) an expected significant increase of diffusive transport if an underlying attenuation mineral layer is not provided . Exothermic degradation of organic matter or hydration of incinerator ash within the landfill generates heat inside the waste pile. This creates thermal gradients through the composite liner, which hold the potential to cause a net movement of moisture away from the warmer liner. The result is a potential for desiccation that may impair the long-term performance of the GCL . As the role of the GCLs broadens, they are being investigated intensively, particularly in respect to their hydraulic and diffusion characteristics, chemical compatibility, mechanical behavior, durability and gas migration -. These methods have proved to be expensive and, in many cases, ineffective at achieving the proposed level of cleanup. The biofilm accumulation in the porous media is an effective way in the reduction of hydraulic conductivity and concentration of organic contaminants from the leachate. This technique stimulates the microbes to remove subsurface pollutants, which is prominent because it has the potential to: permanently eliminate the contaminants through biochemical transformation or mineralization; avoid harsh chemical and physical treatments; operate in situ and be cost effective ,. Aquifer material is excavated and replaced with microbial cultures as horizontal treatment layers and as vertical treatment walls. These engineered biofilm layers eliminate the contaminants on in-situ transformations . Among the various contaminated groundwater remediation measures that consider results, risks, and costs, biofilm accumulation is preferred .
A biofilm is a well organized, cooperating community of microorganisms. Microbial cells attach to the surfaces and develop a biofilm. Biofilm associated cell is differentiated from suspended counterparts by reduced growth rate, upwards and down regulation of gene and generation of extra polymeric substances . Genetic studies have revealed that biofilms are formed through multiple steps. They require intracellular signalling and transcribe different set of genes from planktonic cell . Biofilm accumulation in porous media is the overall result of microbial cell adsorption, desorption, growth on surfaces, detachment and filtration. The microbial growth in land and the resultant decrease in hydraulic conductivity are much connected with groundwater recharge, wastewater, soil injection, enhanced oil recovery schemes, and the in situ bioremediation of organic contaminants in the subsurface environment .
A turn of previous experiments have looked into the issue of microbial biomass growth on reduction of porosity, permeability and hydraulic conductivity in porous media -,,, and rock-fracture analogues , which could induce an outcome on the fate of contaminants in the subsurface.
Seki et al. concluded that bacterial clogging proceeds more rapidly than fungal clogging probably because bacteria grow faster than fungi on the ground surface. Taylor and Jaffe  conducted experiments on sand packed column reactors to study the issue of biomass growth on soil permeability and dispersivity using methanol as a growth substrate. From the answers, the permeability reduction was noted by three orders of magnitude. Vandevivere and Baveye  and Bielefeldt et al. estimated the hydraulic conductivity reductions of three orders of magnitude. Cusack et al. reported that the decrease in K (hydraulic conductivity) was achieved as 99% in sandstone by using starved bacteria. Cunningham et al.  used sand (grain size: 0.54 mm and 0.12 mm) column inoculated with bacteria and applied a constant head difference between inflow and outflow. A reduction of more than 90% in hydraulic conductivity and 50–90% in porosity was observed. Brough et al.  noted a reduction of hydraulic conductivity of between 28 and 79% using 35 series of column experiment.
Kim  evaluated the changes in hydraulic conductivity as 1 × 10-4 cm/Sec is using sand columns (sand grains of 0.25 millimeter to 0.42 mm in size) due to barrier formation. Kim et al.  reported the hydraulic conductivity reduction by 1/8000 of the initial hydraulic conductivity when the uninterrupted provision of substrate and oxygen. Zhong and Wu et al.
Kim  evaluated the changes in hydraulic conductivity as 1 × 10-4 cm/Sec is using sand columns (sand grains of 0.25 millimeter to 0.42 mm in size) due to barrier formation. Kim et al.  reported the hydraulic conductivity reduction by 1/8000 of the initial hydraulic conductivity when the uninterrupted provision of substrate and oxygen. Zhong and Wu et al. investigated the bioclogging in porous media (sand grains of size 0.2 mm to 0.5 mm) under continuous flow condition and achieved the hydraulic conductivity reduction in one order magnitude. Several authors reported a significant reduction of hydraulic conductivity due to bioclogging. These studies demonstrated that the biobarrier may be a promising technology for containing contaminant plume in the field. Improved understanding of these interactions will lead to industrial and environmental applications in bio hydrometallurgy, enhanced oil recovery, and bioremediation of contaminated groundwater and soil .
In this research study, the influence of biofilm accumulation using sand column is described by (1) the reduction in hydraulic conductivity of the inoculated microbial sand column (2) the physico-chemical characteristic of the effluent leachate such as pH, turbidity, total dissolved solids (TDS), oxidation reduction potential (ORP), nitrates, phosphates and degradation of organic contaminants in terms of COD.
Materials and methods
Physical properties of sand
1 × 10‒1
Dry density (g/cm3)
Organic content (%)
Moisture content (%)
All the chemicals used in this study were of analytical reagent (AR) grade and were supplied by Merck specialities Ltd., Mumbai, India. Glassware used for analysis was washed with an acid solution followed by distilled water.
Isolation of microorganism
In this study, bacterial strains were isolated from the leachate samples collected from an open dumping site at Ariyamangalam, Tiruchirappalli, Tamilnadu. To eliminate the target contaminants from the leachate, the experimental microorganisms were isolated from leachate sample according to normal microbiological procedures. The nutrient medium (NM) for bacterial growth consists of Peptone (10 g), Beef extract (2 g), Yeast extract (1 g), and Sodium Chloride (5 g) in 1L of distilled water . The pH was maintained at 7±0.2 through the addition of HCl (0.1 N) or NaOH (0.1 N). The media were sterilized by wet autoclaving at 15 kPa and 121°C for 20 min. Approximately 10 ml of leachate was added to 100 ml of nutrient medium (NM) and incubated for 48 h at 37°C in facultative condition . The shake flask cultures were closed using Teflon stoppers.
The growth rate of the microorganisms was estimated by measuring Optical Density (OD), defined as the logarithmic ratio of the initial light intensity to the light intensity not disturbed by the microorganisms. OD can be measured by using a spectrophotometer at 600 NM wavelengths . A loopful of incubated mixed culture was streaked on agar slants, incubated for 24 h and stored in the freezer at 4°C for further use.
Bacterial cultivation in synthetic leachate
Composition of synthetic leachate
Quantity per litre
Titrate to an Eh 120 mV:180 mV
Titrate to a pH 5.8‒6.0
Trace metal solution (TMS)
To make 1 l
Composition of trace metal solution (TMS)
96% concentration H2SO4
To make 1 l
Physicochemical characteristics of synthetic leachate
Turbidity in NTU
Total Dissolved Solids (TDS) in mg/L
Nitrates (NO3‒) in mg/L
Phosphates (PO43‒) in mg/L
Chemical Oxygen Demand (COD) in mg/L
The cleaned sand of size 0.6 mm and 0.3 mm were used for filling each column. A mixture of 70% of 0.3 mm sand and 30% of 0.6 mm sand, by weight (70:30 mix) were used for filling the first two columns. Column 1 was filled with mixed sand media (MSM); whereas column 2 was amended with microbes in mixed sand media (MSMM). The other two columns were filled with stratified layer of sand media such as, 110 g of 0.3 mm sand in the top layer (0‒15 cm) and 100 g of 0.6 mm sand in the bottom layer (15 ‒ 30 cm). Column 3 was filled with sand media in stratified mode (SSM); whereas column 4 was amended with microbes in stratified sand media (SSMM). Totally, 210 grams of sand media were weighed and packed for a depth of 30 cm of each column. Column 1 and 3 were used as a blank, without any addition or inoculation. To inoculate the columns 2 and 4, 80 ml of developing a culture containing approximately 340 × 105 cells/ml were mixed with 210 g of sterilized sand, and the resulting slurry was aseptically poured into the columns. The inoculated sand columns were incubated under no-flow conditions for 24 h, to promote bacterial attachment to the sand . Glass wools were placed at the outlet of the each column to retain the sand in the columns. The experiments were carried out at a temperature of 20 ± 1°C and the sand-bed columns were side protected from the light .
The synthetic leachate described previously was fed to the inlet tank, which can be located at the preferred height. The overflow was circulated to the inlet tank to maintain a constant hydraulic head. The pH of the synthetic leachate was adjusted as 5.8 to 6.0 throughout the experimental study. A four channel peristaltic pump was set to distribute a constant flow of 1 ml/min synthetic leachate for a combined flow rate of 4 ml/min. volumetric flow rates were monitored frequently. To begin with, the outlet flow rate was also set at 1 ml/min for all the columns. Saturated conditions in the columns were kept by controlling the water surface 5 cm above the sand-bed.
K - hydraulic conductivity (cm/s)
Q - volume of flow (cm3)
L - length specimen (cm)
A - cross sectional specimen area (cm2)
t - time during which Q occurs (s) and
h - hydraulic head (cm)
The effluent samples were collected daily from the outlet port of the each column and the physicochemical parameters such as pH, turbidity, Total Dissolved Solids (TDS), Nitrates (NO3‒), Phosphates (PO43‒), Oxidation Reduction Potential (ORP) and COD were analyzed as per standard methods . All the analyses in this study were repeated two or three times until concordant values were obtained.
After completion of the experimental study, the sand samples were collected from each column and surface morphology has been visualized by Scanning electron microscopy (SEM). Since SEM is important for high resolution visualization of bacterial biofilms . In SEM, biofilm specimens are prepared by fixation, staining, drying and conductively coating prior to imaging under high vacuum . Air dried samples were spread out on the sample mounted on aluminum stab sequenced by coating with a thin layer of gold under vacuum to increase the electron conduction and to increase the quality of the images . The scanning electron imaging of sand samples of each column after leaching was done at 1–15 KV uses a microscope equipped with a filled-emission cathode. The images were captured using SEM HITACHI (Model: S3000N) instrument with 100× and 500× magnification.
Results and discussion
Hydraulic conductivity (K)
After 45 days, the reduction in hydraulic conductivity was observed as 10% in MSM and 7% in SSM. The microbial inoculated column MSMM reduced hydraulic conductivity up to 98.88% during the 45 days of experimental operation. Within 2–3 days after microbial inoculation, a uniform biofilm of detectable thickness could be observed on the exposed edges of reactor media particles. Rapid decrease in K (68%) was attained in the first six days due to hasty microbial growth and its uniform biofilm thickness along the column. Mixed bio culture in the porous media and the uniform biofilm formation are responsible in hydraulic conductivity reduction from 24th day onwards. The uniformity biofilm growth was happening due to the mixed proportion (70:30 mix) of porous sandy media; it allows the microbes to attach with the sand particles and further increases the biofilm growth. Hydraulic conductivity in the stratified media inoculated with microbes achieved a 93.94% reduction after 45 days of operation. For the first 6 days, 66% of reduction in K was observed due to the initial inoculums in the column. Subsequently, 90% of K reduction was achieved on 19th day, due to the biofilm formation. During the period between 27 and 45, the K value stabilized at 93%. The higher reduction in hydraulic conductivity was achieved in MSMM column when compared to SSMM column.
Effluent leachate composition changes
Gradual reduction in SCOD was observed for the day 13 to 22 with less nutrient consumption by the microbes. From the day, 23 to 30 the SCOD concentration turns to the steady state with stable consumption by the microbes. After 30 days of the experiment, the reduction in SCOD concentration in MSMM and SSMM was observed 76.19% and 54.21% respectively. The faster consumption of COD as time elapsed implies that microbial growth was continuous throughout the column depth as nutrient and electron acceptors were continuously provided ,,. When compared to MSMM column, SSMM column achieved a less reduction in the SCOD concentration in the effluent leachate. This may be due to less microbial growth in the coarse sand particles (0.6 mm) of the stratified column.
Values of pseudo-first-order constant for different column conditions
Reaction rate constant k1(day‒1)
Biofilm observation by SEM
The observations from mixed sand media column were made as follows: Figure 10(a, a1) shows an image of the sand surface scan without clogging. The surface of the sand is smooth. Figure 10(b, b1) shows a picture of the surface scan of the sand after 45 days of clogging, which clearly shows the intensive growth of microbial films . The inoculated bacterium forms several layers of mesh forms of biofilm between sand particles as well as on the sand surface. The formation mesh layers effectively clog the sand pore and result in hydraulic conductivity reduction .
The fine sand media were taken from the top of the stratified columns shows that, there was a slight formation of biofilm on the sand surface (Figure 10(c, c1)). This may be due to the less hydraulic conductivity of the fine sand particles used which allows the growth of microbes on the sand surface. Figure 10(d, d1) shows the, clear thick microbial layer on the surface of the sand grain.
The observations of coarse sand samples collected from the stratified column were discussed as follows: From the SEM images it can be seen that there was only a little difference between the blank columns and microbial inoculated columns. Figure 10(e, e1) shows a clear soft surface layer and it was found no microbial growth in the coarse sand media. A very thin layer of biofilm formation visualized on the coarse grain surface of the microbial column (Figure 10(f, f1)). The comparison of fine and coarse sand media concluded that, there was an appreciable formation of biofilm layer occurred only on the fine sand surface.
In this study, the concept of a biofilm accumulation in a sand column for controlling the leachate migration in the subsurface environment is reported. Synthetic leachate was supplied continuously for the microbial growth to measure the reduction in hydraulic conductivity and organic contaminants of the effluent leachate. Of the four different types of column experiment the following conclusions were made: With scanning electron microscope observation of biofilm, it can be observed that the sand column inoculated with bacteria formed several layers of the biofilm on the sand particles, which resulted in a hydraulic conductivity reduction. There was no appreciable change in the hydraulic conductivity and physico-chemical parameters analyzed from the effluent of blank columns (MSM and SSM). The initial hydraulic conductivity of the column was 2.03 × 10‒2 cm/s. After 45 days of operation, 98.8% of hydraulic conductivity reduction was observed in the MSMM column with 2.31 × 10‒4 cm/s. The stratified microbial column (SSMM) reduces the hydraulic conductivity to 1.23 × 10‒3 cm/s with 93.94%. Hence, the mixed microbial sand column achieves the very good reduction in hydraulic conductivity.
The physico-chemical parameters were also implying the good results in the mixed microbial sand column among all four columns. A proper biological activity was observed from the turbidity and ORP results of MSMM column. Additionally, the reduction in nitrates and phosphates were also observed due to uniformity in the growth of microbes and continuous intake by microbes. In the case of the stratified microbial column, less reduction was taken place in the effluent leachate due to the less microbial growth formation in the coarse sand layer. There is a chance of a detachment of microbes in the coarse layer due to the more space between the sand grain particles.
The organic reduction of influent was estimated by SCOD concentration of each column. The column MSMM shows good reduction at 76.19%, whereas in the column SSMM, 54.21% reduction was achieved. This may be due to the continuous intake of organic substrate by the microorganisms. The pseudo-first-order constant value clearly indicates that the degradation of organic contaminants is due to heavy biofilm accumulation inside the sand column. From the overall results, the biobarrier formed by mixed sand media amended with microbes poses a suitable remedial measure for the reduction of hydraulic conductivity and organic contaminants in the leachate.
The first author is extremely grateful to Dr. S. T. Ramesh, Associate Professor for his truly support to carry out this research. All the authors are thankful to National Institute of Technology, Tiruchirappalli, Tamil Nadu for carrying out this work in the Environmental Engineering laboratory.
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