Elsevier

Geochimica et Cosmochimica Acta

Volume 169, 15 November 2015, Pages 30-44
Geochimica et Cosmochimica Acta

Cell surface reactivity of Synechococcus sp. PCC 7002: Implications for metal sorption from seawater

https://doi.org/10.1016/j.gca.2015.07.033Get rights and content

Abstract

The past two decades have seen a significant advancement in our understanding of bacterial surface chemistry and the ability of microbes to bind metals from aqueous solutions. Much of this work has been aimed at benthic, mat-forming species in an effort to model the mechanisms by which microbes may exert control over metal contaminant transport in soils and groundwater. However, there is a distinct paucity of information pertaining to the surface chemistry of marine planktonic species, and their ability to bind trace metals from the ocean’s photic zone. To this end, the surface properties of the cyanobacterium Synechococcus sp. PCC 7002 were studied as this genus is one of the dominant marine phytoplankton, and as such, contributes significantly to metal cycling in the ocean’s photic zone. Zeta potential measurement indicates that the cell surfaces display a net negative charge. This was supported by potentiometric titration and Fourier transform infrared spectroscopy analyses demonstrating that the cells are dominated by surface proton releasing ligands, including carboxyl, phosphoryl and amino functional groups, with a total ligand density of 34.18 ± 1.62 mmol/g (dry biomass). Cd adsorption experiments further reveal that carboxyl groups play a primary role in metal adsorption, with 1.0 g of dry biomass binding an equivalent of 7.05 × 10−5 M of Cd from solution at pH = 8. To put this value into context, in 1 L of seawater, and with an open-ocean population of Synechococcus of 105 cells/mL in the photic zone, approximately 10 nmol of Cd could potentially be adsorbed by the cyanobacteria; an amount equivalent to seawater Cd concentrations. Although we have only focused on one microbial species and one metal cation, and we have not considered trace element assimilation, our results highlight the potential role of surface sorption by phytoplankton in the cycling of metals in the ocean.

Introduction

Bacterial surfaces possess abundant reactive ligands, such as carboxyl, hydroxyl, phosphoryl, and amino groups, that can deprotonate with increasing pH and are capable of binding metal cations (Fein et al., 1997, Cox et al., 1999). Those cations may then serve as sites for the nucleation of authigenic mineral phases (e.g., Clarke et al., 1997, Konhauser et al., 1998). Recently, a number of studies have modeled the proton binding and charge properties of bacterial surfaces to determine the types and abundances of these reactive constituents and the capacity of bacteria to adsorb cations under different environmental conditions (Martinez et al., 2004, Fein et al., 2005, Lalonde et al., 2007, Baker et al., 2010, Kenney and Fein, 2011). Other studies have focused on how microbial surface chemistry and the charge characteristics may influence the microorganism’s hydrophobic and hydrophilic properties (Van Loosdrecht et al., 1987, Mozes et al., 1988, Ahimou et al., 2001), which in turn, affects the ability of a microbe to adhere to solid surfaces (Van Loosdrecht et al., 1987, Ong et al., 1999, Cao et al., 2011). The main motivation for these studies was to better understand the mechanisms by which microbes might inhibit the transport of metal contaminants in the subsurface, and thus, to design effective bioremediation strategies (Bethke and Brady, 2000, Konhauser, 2007).

In the past decade, the database pertaining to cell surface reactivity has increased significantly (Mishra et al., 2010, Kenney and Fein, 2011, Wei et al., 2011). Yet to our knowledge such studies are limited with regards to cyanobacteria, in particular the determination of the organic ligands which contribute to cellular surface charge (Phoenix et al., 2002, Dittrich and Sibler, 2005, Dittrich and Sibler, 2006, Lalonde et al., 2007, Pokrovsky et al., 2008, Hadjoudja et al., 2010) and their role in metal complexation (Benning et al., 2004, Yee et al., 2004, Pokrovsky et al., 2008, Acharya et al., 2009, Acharya et al., 2012, Acharya et al., 2013, Acharya and Apte, 2013). This is somewhat perplexing given that cyanobacteria are found in many terrestrial and aquatic habitats, and as primary producers and nitrogen fixers, they comprise the base of the food chain.

Cyanobacteria can grow either planktonically or benthically, and as such, the different species must have the means to alter their hydrophobic and hydrophilic properties. In this regard, studies have only recently begun characterizing the surface reactivity of cyanobacteria growing as freshwater mats or within hot spring sinters (Phoenix et al., 2002, Dittrich and Sibler, 2005, Lalonde et al., 2007). For instance, Phoenix et al. (2002) showed that the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly anionic cell wall enclosed within a neutrally-charged sheath. Interestingly, the dual-layered distribution of reactive sites on Calothrix sp. has several important ecophysiological implications, one of them being that the cyanobacterial sheath provides a protective mechanism against detrimental biomineralization (Phoenix et al., 2000). In the case of silicification in hot springs, this exopolymer was demonstrated to act as a filter against colloidal silica by restricting precipitation onto the sheath’s outer surface and preventing silicification of the cell wall. Moreover, because Calothrix sp. strain KC97 is a benthic cyanobacterium that inhabits biofilms covering the silica sinters at hot springs, the ability of this microorganism to adhere to a mineral substratum is fundamental at preventing rapid removal from the hot spring site by the relatively fast-flowing discharge waters. In this regard, studies have suggested that bacterial adhesion is proportional to the microorganism’s hydrophobicity and inversely proportional to the bacterium’s surface charge (e.g., Van Loosdrecht et al., 1987).

By contrast, a study of various cyanobacteria by Fattom and Shilo (1984) demonstrated that planktonic cyanobacteria exhibit hydrophilic characteristics. The suggestion that an enclosing sheath of low electronegativity is important in inducing hydrophobicity (and thus encouraging surface adhesion) is corroborated by observations that the free-swimming hormogonia produced by benthic cyanobacteria are all hydrophilic in nature (Fattom and Shilo, 1984). Filaments in this planktonic, transient phase lack extracellular sheaths, thus exposing the highly electronegative cell walls. This, in turn, contributes significantly to the hormogonia’s hydrophilic characteristic. Along similar lines, previous studies confirm that planktonic, Synechococcus-type cells (Dittrich and Sibler, 2005) and Microcystis aeruginosa (Hadjoudja et al., 2010) have low isoelectric points, giving those bacteria a highly negative surface charge and low hydrophobicity.

If planktonic species are hydrophilic, then it follows that those species will also be capable of reacting with polarized water molecules and dissolved metal cations. This has important ramifications for the oceans because the photic zone can be dominated by planktonic cyanobacteria, in particular, the genera Synechococcus and Prochlorococcus. For instance, Synechococcus populations vary from 104 to 105 cells/mL within the photic zone in the relatively rich waters of the Arabian Sea and off the coast of Peru (Waterbury et al., 1979, Worden et al., 2004), while Flombaum et al. (2013) predicted that the average contribution of Synechococcus and Prochlorococcus to ocean net primary production was 16.7% and 8.5%, respectively. Given their abundance in the oceans, understanding their surface reactivity towards cationic trace elements is critical to understanding the role that planktonic cyanobacteria play in trace metal sorption from the marine water column.

In this study, potentiometric titrations and modeling of cell surface reactivity were employed to constrain the acid dissociation constants (pKa) and concentrations of proton-active sites that may bind metals to the cell surface of Synechococcus sp. PCC 7002, while zeta potential and Fourier transform infrared (FTIR) analyses were used to assess the surface chemical characteristics of the marine cyanobacterium. Cadmium adsorption experiments were also performed at various metal:bacterial site concentration ratios as a function of pH, to determine cyanobacterial efficiency at adsorbing Cd from solution, and by extension, other trace metal cations. Cd was specifically chosen in our study versus other metal cations for the following reasons: (1) Cd remains largely soluble at seawater pH; (2) its use in laboratory settings is not complicated by the precipitation of cadmium carbonate or hydroxide solids at our experimental conditions; (3) to facilitate comparison with other metal adsorption studies involving humic acids and other bacterial species where Cd is the usual metal chosen (Borrok et al., 2004a, Borrok et al., 2004b, Yee et al., 2004, Johnson et al., 2007, Ueshima et al., 2008, Alessi and Fein, 2010, Lalonde et al., 2010, Kenney and Fein, 2011, Petrash et al., 2011); and (4) for relevance to estuaries and other near-shore waters where Cd concentrations may be elevated (Petersen et al., 1998).

Section snippets

Bacterial growth

Axenic cultures of the cyanobacterial strain Synechococcus sp. PCC 7002 (henceforth referred to as Synechococcus) were grown at 30 °C in media A (Stevens and Van Baalen, 1973) supplemented with 0.01 M NaNO3 (designated A+ media; Stevens and Porter, 1980) and buffered with 1 M Tris at pH 8.2. Sufficient biomass for potentiometric titrations or Cd adsorptions was obtained by using three 300 mL cultures, inoculated from the same starting culture, and each grown in 1 L Erlenmeyer flasks. Aeration was

TEM and SEM

Fig. 1 shows that Synechococcus sp. PCC 7002 is a small (<1 μm in diameter), sheathless, coccoid cyanobacterium, where the outer membrane acts as a semipermeable barrier toward molecules from the external environment. The internal structure is consistent with other cyanobacteria, containing cyanophycin granules, ribosomes, carboxysomes, thylakoids and a nucleoid region.

FTIR spectra

The functional groups and corresponding infrared spectra collected for Synechococcus are summarized in Fig. 2 and Table 1.

Surface reactivity of planktonic Synechococcus

The primary aim of this study was to characterize the surface reactivity of the sheathless marine cyanobacterium, Synechococcus sp. PCC 7002, by evaluating the ability of organic ligands attached to its cell wall to adsorb metal cations. Since the surface charge of bacterial cells, in general, is established by proton dissociation from surface exposed ligands (Fein et al., 2005), the net electronegativity of Synechococcus cells provides evidence that those ligands are highly reactive for

Acknowledgments

The authors would like to thank the following for their assistance with the various analyses: FTIR in the Department of Chemistry Analytical and Instrumentation Laboratory, University of Alberta (Wayne Moffat); ICP-MS was performed in the Department of Renewable Resources Natural Resources Analytical Laboratory (Xin Zhang); zeta potential measurement was performed in the Department of Chemical and Materials Engineering Laboratory (Jim Skwarok); and SEM and TEM analysis in the Department of

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