Elsevier

Water Research

Volume 44, Issue 4, February 2010, Pages 1279-1287
Water Research

Distribution of aerobic motile and non-motile bacteria within the capillary fringe of silica sand

https://doi.org/10.1016/j.watres.2010.01.001Get rights and content

Abstract

Retention of bacterial cells as “particles” by silica sand during formation of a capillary fringe (CF) and the influence of motility was examined with motile Pseudomonas putida and non-motile Corynebacterium glutamicum suspensions in the absence of nutrients. The fractional retention of C. glutamicum cells at all regions of the CF was higher than for P. putida cells, most probably due to the motility of P. putida. Only about 5% of P. putida cells and almost no C. glutamicum cells reached the upper end of a CF of 10 cm height.

With cell suspensions of P. putida and C. glutamicum in nutrient broth the development of a CF in silica sand fractions of 355–710 μm and 710–1000 μm respectively, was finished after about 6 h. Growth of cells proceeded for about 6 days. P. putida formed a biofilm on silica grains, whereas no attachment of C. glutamicum on silica sand occurred. Relative cell densities of C. glutamicum on the bottom and in the upper regions of the CF were always lower than those of P. putida and were also lower than those reached in suspended cultures with the same medium. In coarse sand the motile P. putida cells reached significantly higher cell densities in upper CF regions than in fine sand. Growth of C. glutamicum in the CF apparently was slower and a higher proportion of the energy was required for maintenance. Whereas cell densities of P. putida, in CFs of both sand fractions, varied less than one order of magnitude, those of C. glutamicum varied in a wider range from the basis to the top of the CF.

Analyses of the esterase activity of P. putida and C. glutamicum with fluorescein diacetate (FDA) revealed that the cells in higher CF regions were significantly more active than those at the bottom of the CF. Furthermore, a significant correlation (r = 0.66, p < 0.01) between cells ml−1 and the FDA conversion to fluorescein was found.

Introduction

The capillary fringe (CF) is a highly variable, commonly oligotrophic natural ecosystem at the transition of vadose zone and groundwater. Structure and extension of CFs are mainly determined by the grain size distribution, wetting of mineral surfaces and surface tension of the aquatic solution (Ronen et al., 1997, Ronen et al., 2000). These determine transport phenomena of gases (e.g. Affek et al., 1998) or solutes and colloids (e.g. Mc Carthy and Johnson, 1993, Abit et al., 2008) through the CF in all directions. Berkowitz et al. (2004) reported that the CF significantly affected water flow and chemical transport from the vadose zone into groundwater, whereas Silliman et al. (2002) demonstrated in laboratory experiments with porous media that water flow and solute transport occurred regularly in the CF in vertical and horizontal direction.

Although many soil scientists and hydrologists have investigated the CF of the vadose zone, the complex interaction of geophysical, geochemical, hydrological and in particular microbiological parameters, such as particle transport or movement and growth of bacteria in the CF has been reviewed by Holden and Fierer (2005), but is not well understood. For investigation of vertical transport of bacteria in water through unsaturated porous media bacterial suspensions were trickled through soil columns (Corapcioglu and Haridas, 1984, Corapcioglu and Haridas, 1985, Schäfer et al., 1998a, Schäfer et al., 1998b; Jewett et al., 1999, Hua et al., 2003, Gargiulo et al., 2007), whereas Trevors et al. (1994) inoculated soil with bacteria and investigated the effect of trickling water on their distribution. Long distance transport of bacteria in sand columns filled with quartz sand under saturated conditions was investigated by Lutterodt et al. (2009). Results of their study indicated a dependency of transport distance from motility, surface hydrophobicity, outer surface potential (Gram negative bacteria) and sticking efficiency. Bacterial transport from the vadose zone into the groundwater is a major concern with regard to public health (Hua et al., 2003, Paul et al., 2004, Xu et al., 2007). Vice versa, migration of Escherichia coli JM109 from the groundwater through the CF into the unsaturated underground was also demonstrated, using a box with porous medium and horizontal water flow and thus simulating the CF at groundwater flow conditions (Dunn et al., 2005). In the studies of Rockhold et al. (2007) a decrease of height and water saturation of a CF in translucent quartz sand by 7–9%, due to colonization by Pseudomonas fluorescens was observed. In own experiments with Hele-Shaw Cells bacteria were transported by capillary forces into the CF of silica sand, but without a superimposed horizontal water flow.

After transport by capillary forces, bacteria need nutrients for growth and biofilm formation in the different zones of the CF. Adsorption coefficients have presumably a major influence on the distribution of bacteria within the sand grain structure of a CF (Camper et al., 1993, Chen et al., 2003). A negative influence on chemotaxis of motile P. fluorescens cells during starvation at low nutrient supply was described by Singh and Arora (2001). Nutrient supply by retention of colloids at air–water–solid interfaces and in zones of immobile water for bacteria to overcome starving conditions was observed by Morales et al. (2009).

The aim of our work was to determine the moisture profile in CFs with different grain sizes, the distribution of aerobic motile and non-motile bacteria during formation of CFs and cell density and activity development with time after the CF was formed. A detailed understanding of activities within the CF could help to improve bioremediation methods and biological filter constructions.

Section snippets

Bacterial strains and media

Two typical soil bacteria, Pseudomonas putida (DSM 291, type strain) and Corynebacterium glutamicum (DSM, 20300, Kinoshita et al., 1958) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ GmbH 2004) and were used in this study. The motile P. putida was cultured in LB medium that contained in g L−1: NaCl, 10; yeast extract, 5 and tryptone, 10; pH 7.0–7.2. The non-motile C. glutamicum was cultured in medium 53 of DSMZ, containing in g L−1: NaCl, 5; yeast extract, 5;

Water saturation and distribution of bacteria within the capillary fringe

The height of capillary fringes (CFs) in Hele-Shaw Cells with silica sand fractions was clearly visible (Fig. 1 B) and varied for silica sand fractions of 355–710 μm and 710–1000 μm only between 9 and 10 cm. Whereas in the first 5 cm above the water level the water saturation decreased only slightly to about 80% there was a more drastic decrease of the water saturation to finally 30–35% at 9 cm height for both grain fractions (Fig. 2).

The transport of resting cells of P. putida and C. glutamicum

Distribution of motile and non-motile bacteria within the capillary fringe

The water saturation in the CF of the smaller silica sand fraction is somewhat higher at the same height than in the CF of the larger sand fraction, but almost identical at the top end. Apparently mainly due to their motility and not directed by nutrient concentrations or chemotactic responses, motile P. putida cells had an advantage to reach the upper end of the CF more numerously than C. glutamicum cells, as judged from the percentage of cells of both organisms that were transported upwards

Conclusions

The following conclusions can be drawn from the capillary fringe (CF) studies:

  • In the silica sand fraction of 355–710 μm grain size the CF reached a height of maximally 10 cm, in the fraction of 710–1000 μm the height was 1–2 cm lower.

  • The water saturation in the CF decreased exponentially with height to 20–30% at the top.

  • In the coarse sand fraction the cell concentration of P. putida was higher at all water saturation levels than in the fine sand faction, whereas the cell concentration of C.

Acknowledgement

This work is part of the DFG research unit DyCap (dynamic capillary fringes) and was financed by DFG, Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg under FOR 831, Wi 524/18-1. We thank D. Bonefas for excellent technical assistance and P. Pfundstein, Laboratorium für Elektronenmikroskopie of the University of Karlsruhe for preparing the SEM microphotographs.

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