Article pubs.acs.org/est
Impact of Dissolved Organic Matter on Bacterial Tactic Motility, Attachment, and Transport Celia Jimenez-Sanchez,† Lukas Y. Wick,‡ Manuel Cantos,† and José-Julio Ortega-Calvo*,† †
Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS-CSIC), Apartado 1052, E-41080-Seville, Spain Helmholtz Centre for Environmental Research - UFZ, Permosertraße 15, D-04318 Leipzig, Germany
‡
S Supporting Information *
ABSTRACT: Bacterial dispersal is a key driver of the ecology of microbial contaminant degradation in soils. This work investigated the role of dissolved organic matter (DOM) in the motility, attachment, and transport of the soil bacterium Pseudomonas putida G7 in saturated porous media. The study is based on the hypothesis that DOM quality is critical to triggering tactic motility and, consequently, affects bacterial transport and dispersal. Sunflower root exudates, humic acids (HA), and the synthetic oleophilic fertilizer S-200 were used as representatives of fresh, weathered, and artificially processed DOM with high nitrogen and phosphorus contents, respectively. We studied DOM levels of 16−130 mg L−1, which are representative of DOM concentrations typically found in agricultural soil pore water. In contrast to its responses to HA and S-200, strain G7 exhibited a tactic behavior toward root exudates, as quantified by chemotaxis assays and single-cell motility observations. All DOM types promoted bacterial transport through sand at high concentrations (∼130 mg L−1). At low DOM concentrations (∼16 mg L−1), the enhancement occurred only in the presence of sunflower root exudates, and this enhancement did not occur with G7 bacteria devoid of flagella. Our results suggest that tactic DOM effectors strongly influence bacterial transport and the interception probability of motile bacteria by collector surfaces.
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coastal environments.8 The environmental fate of organic pollutants can also be affected by DOM via increased apparent solubility, desorption, transport, and biodegradation.9−13 It is known that DOM can act as a mobilizer of bacterial cells via induced changes in the charge of the collector surface or by competitive sorption interactions between adsorbing organic matter and bacteria.14−18 However, knowledge of the physiological effects of DOM on bacterial mobilization is still quite limited. Several studies have found that the tactic responses of bacteria might enhance their transport and dispersal.19−22 Tactic motility, which is observed at the single-cell level as changes in swimming modes, likely influences the deposition of bacteria in saturated porous media. Dissimilar modes of interaction with collector surfaces may result, for example, in reduced cell interception due to smooth movement or in the saturation of collector surfaces due to repellence swimming and hypermotility, which in turn results in differential transport through porous materials.20,21 To the best of our knowledge, no attempts have been made to test whether bacterial tactic responses to DOM contribute to increased bacterial dispersal in porous environments.
INTRODUCTION The transport of bacteria in soil is more than just a physical trait. Indeed, motility and dispersal are key ecological drivers that allow bacteria to move toward niches of beneficial environmental conditions and to maintain key ecosystem functions, e.g., biodegradation of soil contaminants. Bacterial transport also may be influenced by a variety of chemical factors, among which dissolved organic matter (DOM) is likely to play a major role. In environmental sciences, DOM is usually differentiated from particulate organic matter as the size fraction of organic matter smaller than 0.45 μm.1 This fraction normally consists of a multitude of structurally different compounds, all typically present at low concentrations, although it can differ significantly in quality (or biodegradability) and quantity in time and space.2,3 DOM is environmentally relevant as a sensitive indicator of shifts in ecological processes and constitutes a link between several compartments, i.e., biomass, soil, and surface and subsurface water.1 For example, in Europe, the decrease in organic matter content observed in soils has been linked to a concomitant enhancement of DOM content in surface waters.4 Additionally, acid deposition affects the fate and quality of DOM and promotes its transfer from terrestrial to aquatic ecosystems.5,6 Changes in the DOM composition of soil and surface waters influence the dynamics of bacterial communities,7 and DOM can prevent harmful effects in microbiological processes through the attenuation of ultraviolet radiation, especially in freshwater and © XXXX American Chemical Society
Received: November 18, 2014 Revised: February 24, 2015 Accepted: March 3, 2015
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DOI: 10.1021/es5056484 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
(approximately 40 mg L−1 of N and 270 mg L−1 of P). The exact composition of each DOM solution is provided in the Supporting Information (SI) (Table S1). The surface tension of the DOM solutions was determined at 25 °C with a TD1 Lauda ring tensiometer. Cultivation of Bacteria and Preparation of Inocula. The naphthalene-degrading soil bacterium Pseudomonas putida G7 was cultivated and prepared as described elsewhere.20 In brief, the bacteria were grown at 30 °C on a rotary shaker at 150 rpm in Erlenmeyer flasks with 100 mL of MM solution supplemented with 5 mM salicylate. The liquid cultures were centrifuged at 1000g for 10 min, and the pellet was resuspended in either MM or MM supplemented with DOM. Tactic Response. The chemical-in-capillary (CC) method was used to test the tactic response of the cells to DOM. To this end, 0.2 mL of a bacterial suspension with a final optical density (OD600) of 0.02 (106 cell mL−1) was placed in the tactic chamber, as described earlier.20 The cells entering the capillaries after 30 min of incubation were quantified as colony-forming units (CFU) developing on tryptic soy agar. Cell proliferation inside the capillaries with root exudates can be excluded in accordance with the low concentration of C in the DOM solutions. Additionally, no growth was observed in the inoculated culture media with exudates and HA as the sole source of carbon and energy. However, growth was observed with S-200, which could be attributable to the presence of specific C-containing components, very likely fatty acids, which were used by this strain for cell proliferation. Capillaries filled with mineral medium served as controls. The amino acids present in sunflower root exudates were tested at a concentration of 100 mM to ensure the chemotactic reaction. Experimental data are given in triplicate. Significant differences were determined by paired ttests at P = 0.01 (SigmaPlot 8.0, SPSS, Chicago, IL, U.S.A.). The tactic factor was calculated by dividing the CFU value in the presence of DOM and the CFU control value. The tactic response toward DOM was also tested qualitatively with a drop assay.26 To perform the assay, a liquid culture was resuspended in a chemotaxis buffer (100 mM potassium phosphate [pH7.0], 20 mM EDTA) with 1% hydroxypropylmethylcellulose (Sigma Chemical Co.). The viscous cell suspension was layered on the bottom of 60 mm-diameter Petri dishes to a depth of approximately 3 mm. A 10-μL drop of the DOM solution was added to the center of the dish. After 1 h, the development of a ring of turbidity surrounding the drop of DOM was considered evidence of a positive tactic response. Swimming Motility. The swimming behavior of individual cells was determined in cell suspensions, prepared as described above for CC experiments (OD600 of 0.2), with a phase contrast Axioskop 2 Carl Zeiss light microscope (Jena, Germany). The microscope was connected to a Sony Exwave HD video camera (Tokyo, Japan) to characterize bacterial trajectories based on speed and rate of change in direction (RCDI) using the software CellTrak (version 1.5, Motion Analysis Corporation, Santa Rosa, CA, U.S.A.).20 The CellTrak program characterizes single-cell bacterial motility using the values of bacterial position in space (X, Y) over time (5-s videos). The analysis, which involved selecting 10 paths and discarding those with a linear velocity of less than 17 μm s−1, was performed in duplicate for each treatment. Significant differences were determined with ANOVA tests and Tukey HSD, p ≤ 0.01, using the SPSS 11.5 program (SPSS, Chicago, IL, U.S.A.). Bacterial Cell Surface Properties. The surface charge and hydrophobicity of cells resuspended in different DOM solutions
Tactic behavior and biodegradation are strongly connected to microbial functions. Indeed, the chemotaxis receptor genes for many organic chemicals are often found coordinately regulated with the genes that control the metabolism of the same chemicals.23 Therefore, we hypothesized that variable levels of DOM biodegradability may be associated with dissimilar potential to act as chemical effectors of bacterial tactic responses. This could be related to structural features of DOM affecting both microbial assimilation and their role as effectors for chemotaxis. For this reason, sunflower root exudates were selected for this study as a model of fresh and easily biodegradable DOM and soil humic acids (HA) for a processed and less biodegradable type of DOM. The oleophilic fertilizer S200 (a commonly used artificial fertilizer) was also chosen due to the easy assimilation of its C, N, and P sources by microorganisms. Hence, the selection of the three DOM types of DOM was based on their potential to act as biostimulants in bioand phytoremediation measures.11,24,25 The effects of these materials on well-described, naphthalene-degrading, and tactic Pseudomonas putida strains were studied in column percolation systems.16,20,21 Using this approach, we specifically addressed the question of whether the DOM-stimulated tactic response has a role in bacterial deposition under continuous flow conditions. Our results may have implications not only for bacterial transport in biodegradation scenarios, but also for other aspects of environmental microbiology, such as the DOM-stimulated transport of flagellated bacterial species with pathogenic potential.
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MATERIALS AND METHODS Sources of DOM. Root exudates were obtained from sunflower (Helianthus annuus) plants propagated in vitro, as described elsewhere.25 The main organic components are primarily carbohydrates, amino acids, fatty acids, and aromatic acids.25 The exudate DOM fraction was obtained after centrifugation at 31 000g for 3 h.10 Humic acids were isolated from a sandy soil (Typic Humaquept) collected from Doñana National Park in Huelva, Spain, adjacent to Santa Olalla lake (4.3% organic matter, pH 6).3,11,16 This soil supports species of Scirpus, Juncus, and Eragrostis and is a major input of DOM to the lake after heavy rainfall. The 13C NMR-analysis of the HA produced results typical for soil humic substances, i.e., signals indicating carboxylic acid groups (200−170 ppm), aromatic groups (160−100 ppm), and alkyl carbon (50−0 ppm).16 The carboxyl carbon resonances are indicative of the oxidative processes involved in the biodegradation of plant organic matter. The results of elemental analysis of the HA (% w/w ash-free) performed in a LECO CHNS932 elemental analyzer were as follows: C: 47.0%, H: 5.31%, O: 43.72%, and N: 3.97%. The oleophilic fertilizer S-200 was kindly supplied by IEP Europe (Madrid, Spain). This additive is composed of urea and phosphoric esters in a mixture of saturated and unsaturated fatty acids (mainly oleic acid), butoxyethanol, glycol ether and a base for carrying water.24 In all cases, the aqueous matrix in the DOM preparations was an inorganic salt solution (MM) described elsewhere.21 The DOM solutions were used at two different concentrations (determined as total organic carbon, or TOC, by a Shimadzu TOC-V analyzer): one with a range of 123−138 mg L−1 and the other with a range of 13−16 mg L−1. With the exception of S-200 at the high concentration (in which the regular dose of N was doubled), the DOM contributed negligible amounts of N and P to the solutions, which had similar N and P contents B
DOI: 10.1021/es5056484 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Table 1. Influence of Exposure of Pseudomonas putida G7 Cells to Dissolved Organic Matter with Respect to Tactic Response, Attachment to Sand and Deposition during Transport in Saturated Sand Columns tactic responseb a,b
treatment control exudates humic acids S-200
DOM (mg L−1) l
NA 16 ± 0m 129 ± 3 16 ± 1 138 ± 8 13 ± 0m 123 ± 1
b
cell surfaceb
bacterial transport and cell depositionb,c,d
surface tension (mN m−1)
tactic factor
turning eventse
θwf(deg)
ζg(mV)
attachment (%)h
αti
C/C0j
PVk
62 ± 1 65 ± 1 59 ± 1 60 ± 2 62 ± 0m 54 ± 0m *34 ± 0m
1 *2.5 *2 1 0.8 1.2 1.2
51 ± 1 *13 ± 0m *16 ± 7 40 ± 14 44 ± 4 54 ± 17 46 ± 2
47 ± 6 41 ± 6 39 ± 12 44 ± 5 42 ± 7 40 ± 4 47 ± 5
−31 ± 2 −32 ± 2 −32 ± 3 −35 ± 2 −33 ± 4 −28 ± 0m −31 ± 5
38 ± 2 *9 ± 1 *13 ± 1 39 ± 4 *7 ± 0m 41 ± 1 *8 ± 1
0.8 (0.64) 0.05 (0.05) 0.02 (0.06) 0.90 (0.57) 0.06 (0.10) 1.10 (0.51) 0.06 (0.04)
0.05 (0.11) 0.81 (0.81) 0.91 (0.77) 0.03 (0.09) 0.78 (0.68) 0.02 (0.15) 0.77 (0.84)
1.8 (4.2) 2.1 (4.5) 2.6 (4.2) 1.6 (3.3) 2.4 (4.1) 2.2 (4.2) 1.8 (4.2)
Concentration of dissolved organic matter, given as total organic carbon. bValues are reported as mean ± one standard deviation. cExperimental errors are not included for clarity. dFinal experimental values are given in parentheses as an indication of the dynamics of filter blocking. eTotal number of turning events (rate of change of direction greater than 1000° s−1). fθw, contact angle. gζ, zeta potential. hPercentage of cells adhered to sand in batch experiments. iαt , attachment efficiency. jC/C0, transport efficiency. kPV, pore volume. lNot applicable. mStandard deviation
