Approaches for Monitoring Bacterial Distribution

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Chapter 3. Effect of Rhamnolipid and Tergitol Surfactant on the Transport of Rhodococcus erythropolis and Pseudomonas putida in Saturated Sand Columns

Abstract

Enhanced bacteria transport in soil has been extensively studied for use of bioaugmentation processes for in situ bioremediation. Factors such as bacterial hydrophobicity and surfactant can affect the efficiency of bacteria transport in the soil. However, the effect of surfactant (anionic vs. nonionic) modification of the surface of bacteria cells and sand particles on microbial transport in the saturated sand columns has not been studied. This study investigated the effect of rhamnolipid and tergitol on transport behaviours of pulse injected hydrophobic Rhodococcus erythropolis (R. erythropolis) and hydrophilic Pseudomonas putida (P. putida) in a saturated sand column. The influence of the surfactant on the surface hydrophobicity of the bacteria and sand particles was also investigated. The transport of bacteria was gauged by the percentage of bacteria eluted out of the column, and the measures for transport parameters such as dispersion coefficients, retardation coefficients, and first order decay. Surfactant solutions made R. erythropolis and sand particles less hydrophobic while surfactant made P. putida less hydrophilic. Rhamnolipid and tergitol altered bacterial hydrophobicity to a different extent but had an equivalent effect on the hydrophobicity of sand particles. In the absence of surfactant solution, a higher proportion of P. putida (76.7%) was eluted out of the column compared to R. erythropolis (60.3%). Both rhamnolipid and tergitol enhanced the transport of both bacterial strains. A decrease in the values for first order decay was also observed after adding surfactant solution. The addition of surfactant increased the value of Lifshitz-van der Waals and acid-base (LW-AB) interaction energy between bacteria and sand particles. The percentage of bacteria eluted out of the column increased with increasing LW-AB interaction energy between bacteria and sand particles. The first order decay values decreased with increasing LW-AB interaction energy between bacteria and sand particles. The level of LW-AB interaction energy was related to the surface tension parameter for the bacteria. Compared to tergitol, a profound enhancement was observed for rhamnolipid, most likely due to the different LW-AB respective interaction energies between bacteria and sand particles. This study demonstrates the importance of surfactant type and bacterial hydrophobicity in bacteria transport in sand soil systems and provides insights for finding a suitable match between surfactant and bacteria,  particularly when introducing exogenous bacteria to expand the population of indigenous bacteria in contaminated soil during bioremediation.
Keywords: Bacteria Transport; Anionic and nonionic surfactant; Rhodococcus erythropolis; Pseudomonas putida KT2442; LW-AB Interaction energy.

Introduction

In situ bioremediation is relatively cost-effective and environmentally friendly compared to ex situ bioremediation (Mohan et al., 2006). Inoculating contaminated soils with exogenous bacteria is a common approach for stimulating in situ bioremediation. However, bacterial mobility can be slow, with some studies reporting that non-attaching bacteria cannot travel beyond a distance of 1 meter (Q. Li and Logan, 1999; Martin et al., 1996). Several studies have therefore focused on enhancing the mobility of bacteria within soil with the goal of promoting their access to contaminants (Gang Chen et al., 2004; Gang Chen and Zhu, 2004; Gross and Logan, 1995; Pantsyrnaya et al., 2011; Tong et al., 2010). Physical, chemical, biophysical and biochemical factors can affect the transport of bacteria in the soil. Fluid velocity (Hendry et al., 1999), and the solution’s chemistry including pH and ionic strength are examples of physical and chemical parameters. Biophysical and biochemical factors include the cell size and cell surface properties of the bacteria (Abu-Lail and Camesano, 2003; Dong et al., 2002; Gannon et al., 1991; Q. Li and Logan, 1999; Tsuneda et al., 2003), and properties of the clay particles (H. Bai et al., 2016; Bradford et al., 2002) and surfactants (Abu-Lail and Camesano, 2003; H. Bai et al., 2016; Gexin Chen and Walker, 2007; Gang Chen and Zhu, 2004; Tsuneda et al., 2003; Zhong et al., 2016).
Factors such as the hydrophobicity of bacteria and soil/sediment particles, and the use of surfactants, have drawn increasing attention in the study of bacteria transport. Bacteria strains with different hydrophobicity showed different affinity to solid surfaces. For example, hydrophobic bacteria have a stronger affinity to soil particles than hydrophilic bacteria to do so (Gang Chen and Zhu, 2004; Huysman and Verstraete, 1993). Once hydrophilic bacteria have attached to the soil, they would re-suspended at a slow rate (McCaulou et al., 1994) due to their desorption rate, which could lead to a long tail in breakthrough curves (BTCs). The hydrophobicity of soil affected the affinity of bacteria. For example, bacteria tend to attach to a more hydrophobic surface (Gang Chen and Strevett, 2001; Zhong et al., 2016). The use of surfactants (e.g. pentaethylene glycol monododecyl ether (Gang Chen and Zhu, 2004), decaethylene glycol monododecyl ether (Gang Chen and Zhu, 2004) and rhamnolipid (G. Bai et al., 1997; Gang Chen et al., 2004; Zhong et al., 2016) promoted bacteria transport in column systems. These surfactants modified the surface properties of both bacteria and soil/sand particles, thereby altering bacteria transport in the column. However, the effect of anionic and nonionic surfactant on bacteria transport is still unclear. Anionic rhamnolipid and nonionic tergitol may alter the hydrophobicity of R. erythropolis 3586 and hydrophily of P. putida 852 to differing extents (Feng et al., 2013a). Rhamnolipid is thought to remove the lipopolysaccharide from the surface of P. putida 852 (Feng et al., 2013a) and P. aeruginosa (Al-Tahhan et al., 2000) thereby lowering the values of surface tension parameters. The hydrophilic head of tergitol likely interacts with hydrophilic P. putida 852. In the treatment of R. erythropolis 3586 with surfactant solutions, the hydrophobic tail of the surfactant is likely to interact with hydrophobic bacteria. Although the effects of the hydrophobicity of bacteria and soil/sediment particles, and surfactant on microbial transport have been widely investigated, the comparative effects of anionic and nonionic surfactant on the hydrophobicity of bacteria and soil/sediment particles and thus on microbial transport have not been studied.
This study investigated the effect of anionic and nonionic surfactant on the transport of hydrophobic and hydrophilic bacteria in saturated sand columns. A synthetic nonionic surfactant, tergitol, was used for comparison for anionic biosurfactant rhamnolipid. Both rhamnolipid and tergitol are readily biodegradable and environmentally friendly (J.-L. Li and Chen, 2009) and show low toxicity to contaminant-degrading bacteria (J.-L. Li and Chen, 2009; Rothmel et al., 1998; Zhang et al., 1997; Zhu et al., 2013). Gram-positive Rhodococcus erythropolis and Gram-negative Pseudomonas putida were used to represent hydrophobic and hydrophilic bacterial strains in this study. Contact angle measurements were conducted to investigate the surface tension parameters of bacteria and sand particles. A pH of ~7 and ionic strength of 0.01M was used to simulate a typical soil environment. Bacteria transport was assessed by the percentage of bacteria that was eluted out of the columns, the breakthrough curves (BTCs) and the transport parameters. Bacteria transport was interpreted with the Lifshitz-van der Waals and acid-base （LW-AB ）interaction between bacteria and sand particles.

Materials and Method

Materials

Surfactants

Anionic rhamnolipid (JBR 210, JENEIL ® Biosurfactant Co.) was used as the biosurfactant in this work. It is a rhamnose-containing glycolipid surfactant that has been primarily produced by Pseudomonas aeruginosa (Desai and Banat, 1997). The synthetic nonionic surfactant Tergitol 15-S-12 (Sigma–Aldrich) was applied as the comparison for rhamnolipid biosurfactant because they had different extent of effect on the bacterial surface properties (Feng et al., 2013a). Rhamnolipid and tergitol were also chosen because they are readily biodegradable and environmentally friendly (J.-L. Li and Chen, 2009) and show low toxicity to bacteria (J.-L. Li and Chen, 2009; Rothmel et al., 1998; Zhang et al., 1997; Zhu et al., 2013). The surfactants were used at a concentration of 1000 mg/L, which is considerably larger than the critical micelle concentration (CMC) of rhamnolipid (40 mg/L) and tergitol (104 mg/L).

Bacterial strains, incubation conditions and quantification of cell numbers

Bacteria were cultured and harvested in the same manner for each experiment. Pure strains of Rhodococcus erythropolis (R. erythropolis) (New Zealand Reference Culture Collection, ESR, Porirua, New Zealand) and Pseudomonas putida KT2442 (P. putida KT2442) (New Zealand Reference Culture Collection, Medical Section) were used in the study. These two bacterial strains were selected not only because they are commonly found in the natural soil environment, but also because of their different surface properties and ability to degrade organic pollutants (Dennis and Zylstra, 2004; Gottfried et al., 2010; Lang et al., 2016; Trzesicka-Mlynarz and Ward, 1995; Yang et al., 2014; Zhao et al., 2009). The seed stocks of the bacteria strains were prepared by mixing 0.5 mL of an overnight culture with 0.5 mL of sterile 50 % glycerol in a 1 mL sterile plastic tube, and then stored at -80 ℃.
erythropolis: The water contact angle could indicate the hydrophobicity of bacterial surface: it is regarded that the water contact angle of hydrophilic bacteria is less than 45°, vice versa. (Daffonchio et al., 1995; Grotenhuis et al., 1992). R. erythropolis has a hydrophobic surface with a water contact angle of 94.7°. Its optimal growth temperature is 25~30 ℃. A loopful of seed stock of R. erythropolis was taken and placed on tryptic soy (TS) agar plates and then incubated at 28 ℃ for 72 hours until visible colonies formed. To obtain enough bacterial cells, we then transferred R. erythropolis cells to new TS agar plates and incubated at 28 ℃ for 72 hours. At least four plates were prepared each time to ensure a sufficient quantity of cells could be harvested for each experiment. Bacteria cells were collected by using a moistened swab.
putida KT2442: P. putida KT2442 has a water contact angle of 36.2°, indicating a hydrophilic surface. The optimal growth temperature for P. putida KT2442 is 25~30 ℃. A loopful of the seed stock was taken and plated onto a TS agar plate, and placed in an incubator at 28 ℃ for 24 hours. After the bacterial cell colonies had formed, the bacterial agar plate was stored in a refrigerator at 4℃ and kept for two weeks. To prepare bacteria for experiments, a colony of bacteria was taken from the agar plate and incubated overnight in 20 mL TS broth in 100 mL sterile flask at 28 ℃, under constant shaking at 200 rpm. Then 20 mL of the bacteria solution was transferred to 2000 mL of fresh TS broth and incubated overnight at 28 ℃, under constant shaking at 200 rpm. P. putida KT2442 cells were harvested by centrifugation at 8000 g for 10 minutes.
The harvested cells were washed three times with saline to remove soluble extracellular polymeric substance and finally resuspended in elute agent solution for further experiments. The initial concentration for batch and column experiment was 3.5 × 10¹⁰ CFU/mL and 8.00 × 10⁹ CFU/mL, for P.putida KT2442 and R. erythropolis, respectively. The number of bacteria was quantified by measuring the fluorescence signals of collected samples using a Perkin Elmer EnSpire 2300 multilabel plate reader and the Wallace Envision Manager software program. The details of the plate reader and CFU/mL measurements and results are provided in the appendix. A linear relationship was obtained between fluorescence intensity and CFU/mL, with a R² value of 0.9597 (Figure A-1 in the appendix).

Interaction Energy Calculation and Surface Tension Parameters Measurement

This study only considered the LW-AB interactions between bacteria and sand particles because the addition of surfactant solution did not significantly affect the electrophoretic mobility of bacteria. The LW-AB interactions energy ( DG_adh ) can be calculated from the surface tension parameters as follows (Absolom et al., 1983; Busscher et al., 1984; Sharma and Hanumantha Rao, 2002; C. Van Oss, 1995):
where subscript b denotos bacteria, l denotes liquid, s denotes solid.
The three unknown surface free energy components, γ^LW (LW apolar component), γ^– (electron donor), γ⁺ (electron acceptor),in Equation 3-1 and 3-2 can be determined using the LW-AB approach and Yong’s equation (Knox et al., 1993) with the contact angle measurements from three diagnostic liquids with known surface tension components. The contact angle measurement for bacteria followed the method used in a previous study (Feng et al., 2013a) where a homogenous bacterial lawn was prepared and then measured using a digital goniometer by dropping a diagnostic liquid on the bacteria lawn. Since quartz is the major component of silica sand, it also offers a homogeneous surface for estimating the surface hydrophobicity of silica sand. In this study, water contact angle and the free energy of bacteria aggregation were used to indicate the hydrophobicity of bacterial cell surfaces. Details of the contact angle measurements are provided in the appendix. Details of the calculation of the free energy of bacteria aggregation are provided in the appendix for Chapter 3.

Column Set-up and Bacteria Transport

A cylindrical stainless steel column (6.15 cm diameter (d) × 41 cm length (L)) was designed for the column experiments. The column has three aqueous sampling ports, termed Port 1, Port 2 and Port 3. Ports 1, 2 and 3 are located 3.0 cm, 15.5 cm and 30.5 cm from the bottom of the column, respectively (Figure 3-1). For each experiment, the column was packed with 1852.4 g of sterilized sand with particle size ranging from 0.0625 mm to 2 mm. The porosity (θ) of the packed column was 0.23 using gravimetric analysis. The column was pre-saturated by purging with 700 mL of eluent agent (e.g. water, rhamnolipid and tergitol) solution before each experiment. The purging direction was from the bottom to the top of the column as shown in Figure 3-1. After pre-saturation, 120 mL of bacterial suspension was introduced into the column at a flow rate of 3.0 mL min^-1. The column was then eluted with a 700 mL solution of bacteria-free solution. Aqueous samples were collected from the three sampling ports and analyzed for bacteria transport. The effluent was collected up until the end of the experiment to determine the percentage of bacteria that was eluted out of the column. The concentration of bacteria in the each sample was normalized to the initial concentration of bacteria (C₀). The normalized concentration of bacteria (C/C₀) was plotted against the actual pore volumes of eluent liquid agent that passed through the column, which is known as the breakthrough curve (BTC). All BTCs were averaged from two sets of experiments.
The actual pore volumes of eluent liquid agent that passed through the column at time t were calculated:
where q is velocity in mL/min, t is time (minutes), d is the diameter of the column in cm, and θ is the porosity of column.

Mathematical Modeling

The mathematical model used in this study considered the transport of bacteria introduced into the column to be the result of convection and diffusion of the liquid due to the concentration gradient (Gang Chen and Zhu, 2004). We considered the bacteria to be transporting through a uniform, one-dimensional, saturated porous media, in other words at a steady-state flow, according to the one-dimensional convention-dispersion equilibrium equation (CDE) as follows (Van Genuchtenm and ALVES, 1982):
where C is the bacteria concentration, D is the dispersion coefficient (includes both diffusion and hydrodynamic dispersion), t is the time, and v (v=q/ε (LT^-1) is the average velocity inside the column. In this study, the equilibrium equation and nonequilibrium equation (information is provided in the appendix for Chapter 3) were solved by CXTFIT model in STANMOD (a Windows-based software package) (Parker and Van Genuchten, 1984), which has successfully predicted the adsorption and transport of E. coli and P. fluorescens (Gang Chen and Zhu, 2004). The parameters, dispersion coefficient (D), retardation coefficient (R_d), first order deposition coefficient (µ) and the coefficient of determination (R²), were estimated. All modeling was performed at zero initial concentration and zero production.

Results and Discussions

The Modification of Surfactant on the Surface Tension Parameters of Bacteria and Sand Particles

The surface tension parameters of bacteria and sand particles were determined through contact angle measurement to understand the effect of surfactant on their respective surface hydrophobicities, as shown in Table 3-1. The ANOVA test suggests that the addition of different types of surfactant did not have a significant effect on the surface tension parameters (P > 0.05). But, In terms of free energy of aggregation ( G1w1) that rhamnolipid and tergitol increased the value of G_1w1 from -81.6 mJ/m² to – 56.6 mJ/m² and -70.2 mJ/m² (Table3-1). However, the surfactants made P. putida KT2442 less hydrophilic, as indicated by the decrease in the value of G_1w1 in the presence of rhamnolipid and tergitol, respectively (Table 3-1). Our findings are consistent with the study by Feng et al. (2013a), which found surfactant decreased the value of G_1w1 for hydrophilic P. putida 852 and reduced the value of G_1w1 for hydrophobic R. erythropolis 3586. The photomicrographs of R. erythropolis and P. putida KT2442 cell suspension in water and rhamnolipid solution provide a visual representation of the behaviours of the bacteria in the different suspension agents. Hydrophobic R. erythropolis cells were shown as more likely to aggregate in water (Figure 3-2A), but suspended more homogeneously in rhamnolipid solution (Figure 3-2B). No significant difference was observed for the aggregation of P. putida KT2442 in water and rhamnolipid solution (Figure 3-2 C and D). Table 3-1 also shows that rhamnolipid biosurfactant and tergitol increased the free energy aggregation G_2w2 between water and quartz from -9.0 to 30.2 and 30.6 mJ/m². This result is in line with observations by Chen et al. (2004) that the addition of rhamnolipid increased the surface free energy aggregation of silica sand. When comparing the effects of rhamnolipid and tergitol in modifying bacteria hydrophobicity, a greater increase in the hydrophobicity of R. erythropolis was obtained with the addition of rhamnolipid, while a greater decrease in the hydrophilicity of P. putida KT2442 was obtained with the addition of tergitol. For quartz, different surfactant type demonstrated equivalent impact on its surface hydrophobicity. These results indicate that anionic and nonionic surfactant can modify bacterial hydrophobicity to a different extent, but have a similar modification on the hydrophobicity of sand particles.

Comparison of the Transport of R. erythropolis and P. putida KT2442 in the Absence of Surfactant Solution

The transport of hydrophobic R. erythropolis and hydrophilic P. putida KT2442 in the absence of surfactant solution was investigated to assess the effect of the hydrophobicity of bacteria on their transport in the column (Figure 3-3A and Figure 3-4A). Firstly, 60.3 % and 76.7 % of total injected hydrophobic R. erythropolis and hydrophilic P. putida KT2442 respectively were eluted from the column. The peak height of BTCs of both bacteria strains decreased with increased travelling distance (Figure 3-3A and Figure 3-4A). The observed BTCs were fitted by nonequilibrium and equilibrium equation to further understand the bacteria transport behaviour. The value of beta (β) in the nonequilibrium equation suggests that all the BTCs can fit through the equilibrium equation because most of its value reaches 0.9999 (Table A-2 in appendix for Chapter 3). The fitted data from the equilibrium equation showed a good description of breakthrough curves with R² ˃0.9270 (Table 3-2). The fitted parameters (Table 3-2) for the hydrophobic and hydrophilic bacteria showed a similar pattern: the highest value of first order decay and the retardation was always observed at Port 1 and the values then decreased with increased travelling distance. These results are in line with those from a previous study (Gargiulo et al., 2008) where most of bacterial cells were retained close to the inlet of columns and the rate of deposition rapidly decreased with increasing in distance. The value of dispersion coefficient that was observed for the different ports was close, indicating that the column was packed at an acceptable level of homogeneity (G. Huang et al., 2006).
When comparing the fitted transport parameters, R. erythropolis presented a greater value for first order decay than P. putida KT2442 (Table 3-1). For example, the values of the first order decay (μ) for hydrophobic R. erythropolis were 0.030±0.004/min, 0.019±0.001 /min and 0.013±0.001 /min respectively for Port 1, Port 2 and Port 3, while a smaller value of μ was observed for hydrophilic P. putida KT2442 at each port, 0.030±0.005 /min, 0.012±0.001 /min and 0.08±0.001 /min for Port 1, Port 2 and Port 3, respectively. Compared to R. erythropolis, a greater value of retardation coefficient (R_d) was obtained for P. putida KT2442 at each port. For example, R. erythropolis and P. putida KT2442 respective R_d values of 1.08±0.03 and 1.73±0.06 at Port 1. The retarded transport of hydrophilic bacteria is consistent with previous findings (McCaulou et al., 1994) that once hydrophilic bacteria were attached to the soil, they would re-suspend at a slow rate. Similarly, hydrophobic R. erythropolis had a smaller dispersion value with a smaller dispersion coefficient (D) than hydrophilic P. putida KT2442 (Table 3-2). These results indicate that hdyrophilic bacteria are more likely to transport through columns.

Table of Contents
Abstract
Acknowledgments
Table of Contents
List of Figures
List of Tables
List of Acronyms
Chapter 1. General Introduction
1.1. Key Factors Influencing the Efficiency of in situ Bioremediation
1.2. Transport of Bacteria
1.3. Bacteria Transport with Microbubble
1.4. Monitoring Bacteria Transport in Soil
1.5. Research Hypotheses
1.6. Research Objectives
1.7. Thesis Outline
Chapter 2. Literature Review
2.1. Introduction
2.2. Bacteria Transport in Soil
2.3. Microbubble
2.4. Approaches for Monitoring Bacterial Distribution
2.5. Approaches for Bacteria Transport in Porous Media
2.6. Model Simulation for Bacteria Transport
Chapter 3. Effect of Rhamnolipid and Tergitol Surfactant on the Transport of Rhodococcus erythropolis and Pseudomonas putida in Saturated Sand Columns
Abstract
3.1. Introduction
3.2. Materials and Method
3.3. Results and Discussions
3.4. Conclusion
Chapter 4. Upward Transport of Rhodococcus erythropolis and Pseudomonas putida with Continuously Injected Microbubble in Sand Column: the Role of Microbubble Generation Method, Surfactant Type, Bacterial Hydrophobicity and the Porosity of Sand Column
4.1. Introduction
4.2. Materials and Method
4.3. Results and Discussions
4.4. Conclusion
Chapter 5. Employing a Novel Optical Biosensor for in situ Monitoring of Bacteria Transport in Saturated Column in Varying Physicochemical Conditions
5.1. Introduction
5.2. Materials and Methods
5.3. Results and Discussions
5.4. Conclusion
Chapter 6. Conclusions, Implications, and Recommendations for Future Research
6.1. Conclusions and Implications for Bioremediation
6.2. Recommendations for Future Research
Bibliography
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Transport of Bacteria in Column for in situ Bioremediation Application