Elsevier

Chemical Geology

Volume 289, Issues 1–2, 7 October 2011, Pages 48-54
Chemical Geology

Research papers
Comparison of the acid–base reactivity of free-living Pseudomonas putida cells and biofilm

https://doi.org/10.1016/j.chemgeo.2011.07.007Get rights and content

Abstract

The proton reactivity of a soil bacterium, Pseudomonas putida ATCC12633 was investigated in two physiologically different states: (i) as free-living cells, and (ii) as a 5-day biofilm formed in a sandy column.

Acid–base data analysis and modeling based on a three-site non-electrostatic model showed that biofilm has a proton exchange capacity 3.8 times higher than that of free-living cells (13.2 mmol/gprotein corresponding to 5.9 ± 1.2 mmol/gdry weight for biofilm and 3.8 mmol gprotein; 1.56 ± 0.32 mmol/gdry weight for free cells). The higher proton exchange capacity of the biofilm fragments mainly results from the high content of the ‘neutral’ pK 6.5 sites. This increase was explained through sorption on the biofilm of mineral phosphate residues circulating with the nutrient medium during the 5 days of column biofilm growth. SEM biofilm observations show a dense network of excreted organic materials linking the individual cells and organic residues. On the contrary, free cells are clearly individualized. The differences between the morphology and reactivity of biofilms and free cells strongly indicate that the two substrates have different compositions. In opposition to free cells, acidic conditions lead to non reversible biofilm proton exchange properties that could be explained by biofilm coagulation processes. These results show the importance of the growing conditions of biofilm and especially its development on a solid surface in the determination of biofilm composition and reactivity.

Highlights

► We compared the proton exchange property of Pseudomonas putida biofilm and free cells. ► The biofilm was grown in a sandy column under a dynamic nutrient supply. ► A large number of phosphate sites are responsible for the increase of biofilm reactivity. ► Exopolymeric substances may be responsible for the high biofilm reactivity. ► The growth conditions of biofilm influence its proton exchange properties.

Introduction

Bacterial biofilms can play an important role on the mobility of heavy metals in the environment. Aqueous metal surface complexation properties are directly linked to the proton exchange properties of bacterial biofilms that were previously described and modeled (Nealson and Stahl, 1997, Borrok et al., 2005). The proton exchange reactivity of a large number of bacterial strains has been widely investigated. These studies have been performed on both Gram negative and Gram positive strains and some authors have compiled existing data and compared the sorption capacities and reactivities of series of strains. All these authors concluded that the reactivities of different strains are comparable regarding overall acid–base exchange and metal sorption capacity (Fowle and Fein, 1999, Ngwenya et al., 2003, Borrok et al., 2005, Fein, 2006, Guiné et al., 2006, Johnson et al., 2007). Conceptually, chemical equilibrium models representing the reactivity of cell wall components on both Gram+ and Gram− cells were considered. These models were formulated for washed free cell suspensions in closed reactors. This physiological state is not very relevant for bacterial cells in natural environments, as in most natural ecosystems they prefer to grow on interfaces such as biofilms (Beveridge et al., 1997, Nealson and Stahl, 1997, Davies, 2000).

It is known, that the composition of biofilms and free-living cells differs (Costerton, 1999). Microorganisms undergo profound changes during their transition from individualized and rather mobile cells, to cells which are part of a complex, surface-attached community (O'Toole et al., 2000). Biofilms comprise aggregates of living and dead microbial cells within a matrix of extracellular polymeric substances (ExPS) and interstitial voids and channels separating the microcolonies (Costerton, 1999, Pulcini, 2001, Sutherland, 2001, Briones and Raskin, 2003).

ExPS have specific compositions and structures. Their reactivity may therefore vary strongly. The relative amount of other reactive biofilm components such as dead cells may also vary (Beveridge et al., 1997, Flemming and Wingender, 2010). This raises the question as to whether the structural and compositional changes of biofilms are comparable to those of free-living cells. A limited number of publications have focused on this topic. Ueshima et al. (2008) compared Pseudomonas putida free cells and biofilms formed in liquid cultures. They showed that there are no important reactivity changes regarding proton and Cd sorption properties. Tourney et al. (2008) compared ExPS-containing and ExPS-free (enzyme-removed) cells, and highlighted a slight increase in overall reactivity.

Conversely, some authors have pointed out increased reactivity in the presence of ExPS. (Brown and Lester, 1979, Chen et al., 1995, Chen et al., 2000, Guibaud et al., 2003, Black et al., 2004). It thus seems that, in the case of biofilms, it would be more difficult to establish a unique ‘biofilm’ surface reactivity model. Moreover, it is known that biofilm formation may depend on the chemical environment. For instance, Pseudomonas strains produce different types of exopolysaccharides if embedded in soils or in the human body (Marty et al., 1998). Moreover, changes in biofilm composition have been observed with changing growth stage (Sauer et al., 2002), alteration of carbon source (Steinberger and Holden, 2004), water availability (Robertson and Firestone, 1992) and the presence of toxins (for example toluene: Schmitt et al., 1995).

Consequently we decided to study a monospecific P. putida ATCC12633 biofilm grown in a sandy porous matrix under steady state water flow. This allows more faithful simulation of the physico-chemical conditions existing in natural soils and aquifers and comparing the reactivity of free-living P. putida cells. To our knowledge this is the first study comparing the reactivity of free-living cells to a biofilm grown under dynamic conditions in a porous medium.

Section snippets

Cell and biofilm growth and washing

Pseudomonas putida ATCC12633 is a common Gram negative aerobic soil bacterium. Cells initially stored in glycerol at −80 °C were grown in a minimum medium containing 1 g/L sodium acetate, 12.7 mg/L iron sulfate, 11.3 mg/L magnesium sulfate, 111.8 mg/L ammonium chloride and 12.8 mg/L potassium phosphate.

Free cell samples were obtained after 5 days growth in this medium at room temperature, i.e. in the exponential growth phase. The cells were washed three times by centrifugation at 12,000 rpm for 15 min

Proton exchange results

Pseudomonas putida biofilm and free cell titrations differ strongly in curve shape and total sorption capacity (Fig. 1). Between pH 4 and 10 the release in [HS] varies from 2.5 and 10 mmol/gPr (1.03 ± 0.20 and 4.5 ± 0.85 mmol/gDW) for free cells and biofilms respectively. The biofilm sorption capacity is thus about four times higher. The slope of the biofilm curve, and thus the buffering capacity reaches a maximum between pH 6 and 7. This means that acid–base exchange sites with neutral pK values are

Discussion

By applying our 3 pK model, the proton exchange capacity was found to increase from 1.6 to 5.9 mmol/gDW, ranging from free P. putida cells to biofilm developed in sand columns. This corresponds to a 3.8-fold increase of the overall exchange capacity. The reactivity of the free cells fits those commonly observed for free bacterial species. Thus, after compiling a large number of their own and foreign titrations of bacterial species, Borrok et al. (2005) found that the overall mean sorption

Conclusions

In the present study, a monospecific biofilm was grown under dynamic conditions in a sandy column, i.e. under conditions favoring contact with a mineral surface. Thus a biofilm reactivity was obtained which was almost four times higher than that of the free cell compounds. So far such significant differences between free cells and biofilm reactivities have not been observed. We show that these variable reactivities between free cells and biofilm relate primarily to the considerable differences

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