Quantifying bacterial attachment and detachment using leaching solutions of various ionic strengths after bacterial pulse
- Nag-Choul Choi†1,
- Jae-Woo Choi†2,
- Kyu-Sang Kwon3,
- Sang-Gil Lee3 and
- Soonjae Lee3Email authorView ORCID ID profile
© The Author(s) 2017
Received: 28 December 2016
Accepted: 7 February 2017
Published: 14 February 2017
In this study, we quantified the attachment and detachment of bacteria during transport in order to elucidate the contributions of reversible attachment on bacterial breakthrough curves. The first set of breakthrough experiment was performed for a laboratory sand column using leaching solutions of deionized water and mineral salt medium (MSM) of 200 mM with reference to KCl solution by employing Pseudomonas putida as a model bacterium. In the second set of experiment, the ionic strengths of leaching solutions immediately after bacterial pulse were lowered to tenfold and 100-fold diluted system (2 and 20 mM MSM) to focus on the influence of physicochemical factor. Results have shown that bacterial retention occurred in the sand column due to the physical deposition and physicochemical attachment. The physicochemical attachment was attributed to the high ionic strength (200 mM MSM) of leaching solution and the formation of primary energy minimum. Replacing the 200 mM leaching solution with the lower ionic strengths after pulse resulted in the increased tailing of breakthrough curve due to the detachment from the attached bacteria. The detachment could be well explained by DLVO theory, which showed the formation of energy barrier and disappearance of the secondary minimum as the ionic strength gradually decreased. Analysis of mass recovery revealed that 12–20% of the attachment was due to physical and physicochemical attachment, respectively, where the latter consisted of 25–75% of irreversible and reversible attachment respectively.
KeywordsBacteria transport Attachment Reversibility Ionic strength Chemical perturbation DLVO
Transport of bacteria through porous medium has been an important issue for the purpose of bioaugmentation scheme as well as for the prediction of movement of pathogenic microorganisms in aquifer systems. Aquifer systems generally exhibit a low nutrient condition and therefore injection of nutrients medium is often required during application of contaminant-degrading bacteria in order to sustain the bacterial population. It was found that nutrient limitation affected substantially the transport of bacteria through sand (Priestley et al. 2006). In the study of bacteria transport (Choi et al. 2007), mineral salt medium (MSM) with a rather high ionic strength was used to maintain the cell density of the injected bacterial solution during transport.
Numerous studies have shown that bacteria transport are dependent on ionic strength of leaching solutions, and that higher ionic strength led to higher irreversible attachment (Mills et al. 1994), collision (Abramson and Brown 2007; Jewett et al. 1995; Li and Logan 1999) or sticking efficiency (Bolster et al. 2001), and lower mass recovery (Choi et al. 2007; Gannon et al. 1991; Kim et al. 2009a) or deposition rate (Chen and Zhu 2004; Chen and Walker 2007; Kim et al. 2009a, b; Kuznar and Elimelech 2004; Redman et al. 2004; Rijnaarts et al. 1996), all of which may result in an unfavorable condition for bioaugmentation scheme. The occurrence of effluent with lower cell density when leaching solutions with high ionic strength were used is attributed to the formation of the primary energy minimum near the particle surface as a result of compressed electrical double layer, and thereby domination of the London-van der Waals attractive force over the bacteria-surface interaction energies at all separation distances.
One of the methods to overcome the bacterial loss was to apply leaching solution with low ionic strength in the column tests (Gannon et al. 1991; Redman et al. 2004). They found that the decrease in the ionic strength of the pore fluid—thereby eliminating the secondary energy minimum—resulted in the release of the majority of previously deposited bacteria. However, their findings were limited to the investigation on the existence of the secondary energy minimum since they applied the leaching solution with low ionic strength after a complete bacterial breakthrough—a rather long elution after breakthrough—in the column tests. More detailed information on the separation of the primary energy minimum from the secondary energy minimum and the reversible portion of bacterial attachment requires a careful design of breakthrough experiment for a given bacterium and porous medium. One of the solutions to this problem would be to apply leaching solutions with various ionic strengths immediately after a bacterial pulse since the input of lower ionic strength will generate various situations in the interaction energy between bacteria and particle surfaces as leaching solutions propagate down the column system. The objective of this study is to investigate the reversibility of bacterial attachment during the transport through aquifer material and the effect of the chemical perturbation on the bacterial detachment. We conducted bacterial transport experiments by applying leaching solutions of various ionic strengths after bacterial pulse. The bacterial attachment and detachment were quantified through comparison of mass recoveries of bacterial breakthrough curves.
Materials and methods
Organisms and culture preparation
Benzene-degrading bacteria, Pseudomonas putida KCTC-1769, was obtained from Korean Center for Type Cultures, Seoul, South Korea. Bacterial culture was prepared as described previously (Kim et al. 2005). Initially, the bacteria in a freeze-dried state were revived in 250 ml Erlenmeyer flasks containing 100 ml of LB medium over a period of 2 days. One milliliter of culture was transferred to a volume of 500 ml LB broth and incubated at a 30 °C temperature in a 140 rpm orbital shaker. Cells were harvested in the late exponential growth phase by centrifugation.
For column experiments, the cells were washed two times and suspended in deionized water or mineral salt medium (MSM) with pH of 7 and with ionic strength of 200 mM containing following constituents per liter of distilled water: K2HPO4, 6 g; KH2PO4, 4 g; (NH4)2SO4, 2 g; MgCl2, 6.6 g; CaCl2, 2.5 g; ZnSO4, 0.8 g; NiSO4, 0.24 g; (NH4)6Mo7O24, 0.18 g; CuSO4, 0.03 g; MnSO4, 0.5 g; CoCl2, 0.19 g; FeCl2, 0.06 g (Kim et al. 2005), and adjusted to an optical density of 0.5 at 600 nm (OD600). For the surface potential measurement, cells were washed and suspended with 200 mM MSM and 10-, 100-, 1000-fold diluted MSM. All glassware and materials were washed and sterilized in the autoclave at 121 °C for 15 min to prevent any influence by other microorganisms. Effective diameter and zeta potential of cell were determined by surface potential analysis.
Sand materials (Jumunjin silica, South Korea) were used for column tests. Prior to use, mechanical sieving was performed using US standard sieves (Fisher Scientific), No. 30 and 10 so that sand fractions (0.6 mm < ϕ < 2.0 mm) could be retained. After sieving, the quartz sands were washed using deionized water to remove any microcolloids which can interrupt the measurement of optical density of effluent samples, and then were autoclaved for 20 min in 121 lb pressure and oven-dried at 70 °C for 3–5 days. Previous study (Choi et al. 2007) revealed that the sand materials mainly consisted of quartz. The zeta potential of sand material were determined by surface potential analysis.
Surface potential analysis
Electrophoretic mobility and zeta potential of Pseudomonas putida and quartz sand
Zeta potential (mV)
Zeta potential (mV)
Details of experimental conditions imposed on the column experiments
Conditions for pulse input
IS (mM) of leaching solution
Type of leaching solution
Surface interaction energy
Effect of leaching solution on bacterial BTC
Peak concentration, mass recovery (MR) and mass loss (ML) of BTCs of Exp. A, B, C
C Peak a
Effect of ionic strength on bacterial BTC
Bacterial detachment and DLVO theory
In the previous section, the bacterial concentrations in the tailing part were found to increase as the ionic strength was lowered. It is, however, questionable to predict the cause of the increased bacterial mass as a result of decreased ionic strength. Redman et al. (2004) reported that the increased bacterial mass was due to bacteria detachment upon the application of lower ionic strength. They found that bacterial attachment and detachment are partially reversible depending on the ionic strength of leaching solution.
The interaction energy calculated could be related to the effect of ionic strength on the bacterial BTCs, which were shown in Fig. 4. The peak attenuation of bacterial BTCs in the stage 1 can be explained by bacterial attachment onto the primary energy minimum developed in the high range (200 mM) of ionic strength. Since sufficiently high concentration of bacteria was supplied, a large amount of cells is attached in the sand surface. Identical BTCs observed in the stage 2 can be explained by the fact that the range of ionic strength in pore water is too high to experience energy barrier or secondary energy minimum. In this range, the attached bacteria are not detached. In the stage 3, the tailings of the BTCs of Exp. C2 and C3 appeared. This is attributed to the development of the secondary energy minimum. In this condition, some part of the bacteria attached by the primary energy minimum was detached and thus contributes to the reversible attachment. In the stage 4, detachment vanishes as the secondary energy minimum disappears and accordingly almost no difference in the BTCs was found.
Quantification of bacteria attachment and detachment
And thus, the fraction of irreversible physicochemical attachment can be estimated as A ch,irr = 5%. So the physicochemical attachment consisted of 3/4 and 1/4 of reversible and irreversible fraction respectively. In summary, in the high ionic strength condition, about 12 and 20% of the bacterial attachment occurred during transport by physical deposition and physicochemical attachment respectively; where among them, 5 and 15% of cells were attached reversibly respectively.
mineral salt medium
NC carried out the transport experiments, participated in the reference research and drafted the manuscript. JW participated in the design of the study, and supervised the research work. SG carried out the surface potential analysis. KS helped to review and to edit the manuscript. SJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
This subject is supported by Korea Ministry of Environment (MOE) as “GAIA (Geo-Advanced Innovative Action) Project (ARQ201502032001)”. We also thank professor Dong-Ju Kim’s insightful suggestions. Corresponding author acknowledged Korea University for support the establishment of laboratory.
The authors declare that they have no competing interests.
Availability of data and materials
We conducted experiments and data generated. All data is shown in graphs.
The datasets supporting the conclusions of this article are included within the article and its additional files.
Consent for publication
This article does not contain any individual person’s data in any form. “Not applicable” in this section.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
We would like to acknowledge that this work was funded in part by Korea Ministry of Environment (MOE) (ARQ201502032001). This work was also supported by Korea University (K1609961).
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