Shape-Selective Supramolecular Capsules for Actinide Precipitation and Separation.

Improving actinide separations is key to reducing barriers to medical and industrial actinide isotope production and to addressing the challenges associated with the reprocessing of spent nuclear fuel. Here, we report the first example of a supramolecular anion recognition process that can achieve this goal. We have designed a preorganized triamidoarene receptor that induces quantitative precipitation of the early actinides Th(IV), Np(IV), and Pu(IV) from industrially relevant conditions through the formation of self-assembled hydrogen-bonded capsules. Selectivity over the later An(III) elements is shown through modulation of the nitric acid concentration, and no precipitation of actinyl or transition-metal ions occurs. The Np, Pu, and Am precipitates were characterized structurally by single-crystal X-ray diffraction and reveal shape specificity of the internal hydrogen-bonding array for the encapsulated hexanitratometalates. This work complements ion-exchange resins for 5f-element separations and illustrates the significant potential of supramolecular separation methods that target anionic actinide species.

Selective separation of light rare-earth elements by supramolecular encapsulation and precipitation.

Supramolecular chemical strategies for Rare Earth (RE) element separations are emerging which amplify the small changes in properties across the series to bias selectivity in extraction or precipitation. These advances are important as the REs are crucial to modern technologies yet their extraction, separation, and recycling using conventional techniques remain challenging. We report here a pre-organised triamidoarene platform which, under acidic, biphasic conditions, uniquely and selectively precipitates light RE nitratometalates as supramolecular capsules. The capsules exhibit both intra- and intermolecular hydrogen bonds that dictate selectivity, promote precipitation, and facilitate the straightforward release of the RE and recycling of the receptor. This work provides a self-assembly route to metal separations that exploits size and shape complementarity and has the potential to integrate into conventional processes due to its compatibility with acidic metal feed streams.

Tuneable separation of gold by selective precipitation using a simple and recyclable diamide.

The efficient separation of metals from ores and secondary sources such as electronic waste is necessary to realising circularity in metal supply. Precipitation processes are increasingly popular and are reliant on designing and understanding chemical recognition to achieve selectivity. Here we show that a simple tertiary diamide precipitates gold selectively from aqueous acidic solutions, including from aqua regia solutions of electronic waste. The X-ray crystal structure of the precipitate displays an infinite chain of diamide cations interleaved with tetrachloridoaurate. Gold is released from the precipitate on contact with water, enabling ligand recycling. The diamide is highly selective, with its addition to 29 metals in 2 M HCl resulting in 70% gold uptake and minimal removal of other metals. At 6 M HCl, complete collection of gold, iron, tin, and platinum occurs, demonstrating that adaptable selective metal precipitation is controlled by just one variable. This discovery could be exploited in metal refining and recycling processes due to its tuneable selectivity under different leaching conditions, the avoidance of organic solvents inherent to biphasic extraction, and the straightforward recycling of the precipitant.

Role of plant growth promoting bacteria in driving speciation gradients across soil-rhizosphere-plant interfaces in zinc-contaminated soils.

Inoculation of soil or seeds with plant growth promoting bacteria ameliorates metal toxicity to plants by changing metal speciation in plant tissues but the exact location of these changes remains unknown. Knowing where the changes occur is a critical first step to establish whether metal speciation changes are driven by microbial metabolism or by plant responses. Since bacteria concentrate in the rhizosphere, we hypothesised steep changes in metal speciation across the rhizosphere. We tested this by comparing speciation of zinc (Zn) in roots of Brassica juncea plants grown in soil contaminated with 600 mg kg-1 of Zn with that of bulk and rhizospheric soil using synchrotron X-ray absorption spectroscopy (XAS). Seeds were either uninoculated or inoculated with Rhizobium leguminosarum bv. trifolii and Zn was supplied in the form of sulfide (ZnS nanoparticles) and sulfate (ZnSO4). Consistent with previous studies, Zn toxicity, as assessed by plant growth parameters, was alleviated in B. juncea inoculated with Rhizobium leguminosarum. XAS results showed that in both ZnS and ZnSO4 treatments, the most significant changes in speciation occurred between the rhizosphere and the root, and involved an increase in the proportion of organic acids and thiol complexes. In ZnS treatments, Zn phytate and Zn citrate were the dominant organic acid complexes, whilst Zn histidine also appeared in roots exposed to ZnSO4. Inoculation with bacteria was associated with the appearance of Zn cysteine and Zn formate in roots, suggesting that these two forms are driven by bacterial metabolism. In contrast, Zn complexation with phytate, citrate and histidine is attributed to plant responses, perhaps in the form of exudates, some with long range influence into the bulk soil, leading to shallower speciation gradients.

An Ionic Limit to Life in the Deep Subsurface.

The physical and chemical factors that can limit or prevent microbial growth in the deep subsurface are not well defined. Brines from an evaporite sequence were sampled in the Boulby Mine, United Kingdom between 800 and 1300 m depth. Ionic, hydrogen and oxygen isotopic composition were used to identify two brine sources, an aquifer situated in strata overlying the mine, and another ambiguous source distinct from the regional groundwater. The ability of the brines to support microbial replication was tested with culturing experiments using a diversity of inocula. The examined brines were found to be permissive for growth, except one. Testing this brine's physicochemical properties showed it to have low water activity and to be chaotropic, which we attribute to the high concentration of magnesium and chloride ions. Metagenomic sequencing of the brines that supported growth showed their microbial communities to be similar to each other and comparable to those found in other hypersaline environments. These data show that solutions high in dissolved ions can shape the microbial diversity of the continental deep subsurface biosphere. Furthermore, under certain circumstances, complex brines can establish a hard limit to microbial replication in the deep biosphere, highlighting the potential for subsurface uninhabitable aqueous environments at depths far shallower than a geothermally-defined limit to life.

Metal internalization by bacterial cells depends on metal biotoxicity and metal to biomass ratio.

The traditional view of metal adsorption to bacterial surfaces is that it can act as a protective mechanism by externalizing the metal outside the cell. However, numerous studies focussing on the biodynamics of metal uptake using biotic ligand models consider metal adsorption to cell surfaces as an important first step in metal uptake and internalization. In order to resolve these conflicting views, we adsorbed two metals (copper and cadmium) with contrasting metal biotoxicity on E. coli JM109, and quantified the distribution of each metal amongst surface sites, periplasmic space and the cytoplasm. Distribution of each metal depended on biotoxicity and metal to biomass ratio. For both metals, low metal to biomass ratio led to most of the metal being associated with the periplasmic space, with less Cd being taken up by cells overall. At high metal to biomass ratios, most of the Cd was associated with surface sites, whereas Cu also increased in surface sites but remained below periplasmic concentrations. These observations are consistent with metal internalization being the dominant process at low metal to biomass ratios, whereas was active efflux when metal to biomass was high, leading to equilibrium between cytoplasm and surface concentrations. Significantly, efflux was more intense for high biotoxicity Cd, consistent with active enzymatic regulation of Cu internalization/homeastasis, which is essential at low concentrations. Moreover, metal internalization increases as surface-bound metal increases, the maximum being constrained by maximum adsorption consistent with Langmuir adsorption behaviour. SUMMARIZE OF PAPER: Bacterial metal internalization is a function of metal biotoxicity and metal loading.

Soil Bacteria Override Speciation Effects on Zinc Phytotoxicity in Zinc-Contaminated Soils.

The effects of zinc (Zn) speciation on plant growth in Zn-contaminated soil in the presence of bacteria are unknown but are critical to our understanding of metal biodynamics in the rhizosphere where bacteria are abundant. A 6-week pot experiment investigated the effects of two plant growth promoting bacteria (PGPB), Rhizobium leguminosarum and Pseudomonas brassicacearum, on Zn accumulation and speciation in Brassica juncea grown in soil amended with 600 mg kg-1 elemental Zn as three Zn species: soluble ZnSO4 and nanoparticles of ZnO and ZnS. Measures of plant growth were higher across all Zn treatments inoculated with PGPB compared to uninoculated controls, but Zn species effects were not significant. Transmission electron microscopy identified dense particles in the epidermis and intracellular spaces in roots, suggesting Zn uptake in both dissolved and particulate forms. X-ray absorption near-edge structure (XANES) analysis of roots revealed differences in Zn speciation between treatments. Uninoculated plants exposed to ZnSO4 contained Zn predominantly in the form of Zn phytate (35%) and Zn polygalacturonate (30%), whereas Zn cysteine (57%) and Zn polygalacturonate (37%) dominated in roots exposed to ZnO nanoparticles. Inoculation with PGPB increased (>50%) the proportion of Zn cysteine under all Zn treatments, suggesting Zn coordination with cysteine as the predominant mechanism of Zn toxicity reduction by PGPB. Using this approach, we show, for the first time, that although speciation is important, the presence of rhizospheric bacteria completely overrides speciation effects such that most of the Zn in plant tissue exists as complexes other than the original form.

16S rRNA gene metabarcoding and TEM reveals different ecological strategies within the genus Neogloboquadrina (planktonic foraminifer).

Uncovering the complexities of trophic and metabolic interactions among microorganisms is essential for the understanding of marine biogeochemical cycling and modelling climate-driven ecosystem shifts. High-throughput DNA sequencing methods provide valuable tools for examining these complex interactions, although this remains challenging, as many microorganisms are difficult to isolate, identify and culture. We use two species of planktonic foraminifera from the climatically susceptible, palaeoceanographically important genus Neogloboquadrina, as ideal test microorganisms for the application of 16S rRNA gene metabarcoding. Neogloboquadrina dutertrei and Neogloboquadrina incompta were collected from the California Current and subjected to either 16S rRNA gene metabarcoding, fluorescence microscopy, or transmission electron microscopy (TEM) to investigate their species-specific trophic interactions and potential symbiotic associations. 53-99% of 16S rRNA gene sequences recovered from two specimens of N. dutertrei were assigned to a single operational taxonomic unit (OTU) from a chloroplast of the phylum Stramenopile. TEM observations confirmed the presence of numerous intact coccoid algae within the host cell, consistent with algal symbionts. Based on sequence data and observed ultrastructure, we taxonomically assign the putative algal symbionts to Pelagophyceae and not Chrysophyceae, as previously reported in this species. In addition, our data shows that N. dutertrei feeds on protists within particulate organic matter (POM), but not on bacteria as a major food source. In total contrast, of OTUs recovered from three N. incompta specimens, 83-95% were assigned to bacterial classes Alteromonadales and Vibrionales of the order Gammaproteobacteria. TEM demonstrates that these bacteria are a food source, not putative symbionts. Contrary to the current view that non-spinose foraminifera are predominantly herbivorous, neither N. dutertrei nor N. incompta contained significant numbers of phytoplankton OTUs. We present an alternative view of their trophic interactions and discuss these results within the context of modelling global planktonic foraminiferal abundances in response to high-latitude climate change.

Mixed planting with a leguminous plant outperforms bacteria in promoting growth of a metal remediating plant through histidine synthesis.

The effectiveness of plant growth promoting bacteria (PGPB) in improving metal phytoremediation is still limited by stunted plant growth under high soil metal concentrations. Meanwhile, mixed planting with leguminous plants is known to improve yield in nutrient deficient soils but the use of a metal tolerant legume to enhance metal tolerance of a phytoremediator has not been explored. We compared the use of Pseudomonas brassicacearum, Rhizobium leguminosarum, and the metal tolerant leguminous plant Vicia sativa to promote the growth of Brassica juncea in soil contaminated with 400 mg Zn kg(-1), and used synchrotron based microfocus X-ray absorption spectroscopy to probe Zn speciation in plant roots. B. juncea grew better when planted with V. sativa than when inoculated with PGPB. By combining PGPB with mixed planting, B. juncea recovered full growth while also achieving soil remediation efficiency of >75%, the maximum ever demonstrated for B. juncea. µXANES analysis of V. sativa suggested possible root exudation of the Zn chelates histidine and cysteine were responsible for reducing Zn toxicity. We propose the exploration of a legume-assisted-phytoremediation system as a more effective alternative to PGPB for Zn bioremediation.

Bacteria-zinc co-localization implicates enhanced synthesis of cysteine-rich peptides in zinc detoxification when Brassica juncea is inoculated with Rhizobium leguminosarum.

Some plant growth promoting bacteria (PGPB) are enigmatic in enhancing plant growth in the face of increased metal accumulation in plants. Since most PGPB colonize the plant root epidermis, we hypothesized that PGPB confer tolerance to metals through changes in speciation at the root epidermis. We employed a novel combination of fluorophore-based confocal laser scanning microscopic imaging and synchrotron based microscopic X-ray fluorescence mapping with X-ray absorption spectroscopy to characterize bacterial localization, zinc (Zn) distribution and speciation in the roots of Brassica juncea grown in Zn contaminated media (400 mg kg(-1) Zn) with the endophytic Pseudomonas brassicacearum and rhizospheric Rhizobium leguminosarum. PGPB enhanced epidermal Zn sequestration relative to PGBP-free controls while the extent of endophytic accumulation depended on the colonization mode of each PGBP. Increased root accumulation of Zn and increased tolerance to Zn was associated predominantly with R. leguminosarum and was likely due to the coordination of Zn with cysteine-rich peptides in the root endodermis, suggesting enhanced synthesis of phytochelatins or glutathione. Our mechanistic model of enhanced Zn accumulation and detoxification in plants inoculated with R. leguminosarum has particular relevance to PGPB enhanced phytoremediation of soils contaminated through mining and oxidation of sulphur-bearing Zn minerals or engineered nanomaterials such as ZnS.

Transport and viability of Escherichia coli cells in clean and iron oxide coated sand following coating with silver nanoparticles.

A mechanistic understanding of processes controlling the transport and viability of bacteria in porous media is critical for designing in situ bioremediation and microbiological water decontamination programs. We investigated the combined influence of coating sand with iron oxide and silver nanoparticles on the transport and viability of Escherichia coli cells under saturated conditions. Results showed that iron oxide coatings increase cell deposition which was generally reversed by silver nanoparticle coatings in the early stages of injection. These observations are consistent with short-term, particle surface charge controls on bacteria transport, where a negatively charged surface induced by silver nanoparticles reverses the positive charge due to iron oxide coatings, but columns eventually recovered irreversible cell deposition. Silver nanoparticle coatings significantly increased cell inactivation during transit through the columns. However, when viability data is normalised to volume throughput, only a small improvement in cell inactivation is observed for silver nanoparticle coated sands relative to iron oxide coating alone. This counterintuitive result underscores the importance of net surface charge in controlling cell transport and inactivation and implies that the extra cost for implementing silver nanoparticle coatings on porous beds coated with iron oxides may not be justified in designing point of use water filters in low income countries.

Use of bioreporters and deletion mutants reveals ionic silver and ROS to be equally important in silver nanotoxicity.

The mechanism of antibacterial action of silver nanoparticles (AgNp) was investigated by employing a combination of microbiology and geochemical approaches to contribute to the realistic assessment of nanotoxicity. Our studies showed that suspending AgNp in media with different levels of chloride relevant to environmental conditions produced low levels of ionic silver thereby suggesting that dissolution of silver ions from nanoparticulate surface could not be the sole mechanism of toxicity. An Escherichia coli based bioreporter strain responsive to silver ions together with mutant strains of E. coli lacking specific protective systems were tested against AgNp. Deletion mutants lacking silver ion efflux systems and resistance mechanisms against oxidative stress showed an increased sensitivity to AgNp. However, the bioreporter did not respond to silver nanoparticles. Our results suggest that oxidative stress is a major toxicity mechanism and that this is at least partially associated with ionic silver, but that bulk dissolution of silver into the medium is not sufficient to account for the observed effects. Chloride ions do not appear to offer significant protection, indicating that chloride in receiving waters will not necessarily protect environmental bacteria from the toxic effects of nanoparticles in effluents.

Mechanisms behind bacteria induced plant growth promotion and Zn accumulation in Brassica juncea.

The growth and metal-extraction efficiency of plants exposed to toxic metals has been reported to be enhanced by inoculating plants with certain bacteria but the mechanisms behind this process remain unclear. We report results from glasshouse experiments on Brassica juncea plants exposed to 400mgZnkg(-1) that investigated the abilities of Pseudomonas brassicacearum and Rhizobium leguminosarum to promote growth, coupled with synchrotron based µXANES analysis to probe Zn speciation in the plant roots. P. brassicacearum exhibited the poorest plant growth promoting ability, while R. leguminosarum alone and in combination with P. brassicacearum enhanced plant growth and Zn phytoextraction. Reduced growth in un-inoculated plants was attributed to accumulation of Zn oxalate and Zn sulfate in roots. In plants inoculated with P. brassicacearum the high concentration of Zn polygalacturonic acid in the root may be responsible for the stunted growth and reduced Zn phytoextraction. The improved growth and increased metal accumulation observed in plants inoculated with R. leguminosarum and in combination with P. brassicacearum was attributed to the storage of Zn in the form of Zn phytate and Zn cysteine in the root. When combined with the observation that both bacteria do not statistically improve B. juncea growth in the absence of Zn, this work suggests that bacteria-induced metal chelation is the key mechanism of plant growth promoting bacteria in toxicity attenuation and microbial-assisted phytoremediation.

Self-preservation strategies during bacterial biomineralization with reference to hydrozincite and implications for fossilization of bacteria.

The induction of mineralization by microbes has been widely demonstrated but whether induced biomineralization leads to distinct morphologies indicative of microbial involvement remains an open question. For calcium carbonate, evidence suggests that microbial induction enhances sphere formation, but the mechanisms involved and the role of microbial surfaces are unknown. Here, we describe hydrozincite biominerals from Sardinia, Italy, which apparently start life as smooth globules on cyanobacterial filaments, and evolve to spheroidal aggregates consisting of nanoplates. Complementary laboratory experiments suggest that organic compounds are critical to produce this morphology, possibly by inducing aggregation of nanoscopic crystals or nucleation within organic globules produced by metabolizing cells. These observations suggest that production of extracellular polymeric substances by microbes may constitute an effective mechanism to enhance formation of porous spheroids that minimize cell entombment while also maintaining metabolite exchange. However, the high porosity arising from aggregation-based crystal growth probably facilitates rapid oxidation of entombed cells, reducing their potential to be fossilized.

Carbonate precipitation under pressure for bioengineering in the anaerobic subsurface via denitrification.

A number of bioengineering techniques are being developed using microbially catalyzed hydrolysis of urea to precipitate calcium carbonate for soil and sand strengthening in the subsurface. In this study, we evaluate denitrification as an alternative microbial metabolism to induce carbonate precipitation for bioengineering under anaerobic conditions and at high pressure. In anaerobic batch culture, the halophile Halomonas halodenitrificans is shown to be able to precipitate calcium carbonate at high salinity and at a pressure of 8 MPa, with results comparable to those observed when grown at ambient pressure. A larger scale proof-of-concept experiment shows that, as well as sand, coarse gravel can also be cemented with calcium carbonate using this technique. Possible practical applications in the subsurface are discussed, including sealing of improperly abandoned wells and remediation of hydraulic fracturing during shale gas extraction.

Thermodynamic and kinetic controls on cotransport of Pantoea agglomerans cells and Zn through clean and iron oxide coated sand columns.

Recent observations that subsurface bacteria quickly adsorb metal contaminants raise concerns that they may enhance metal transport, given the high mobility of bacteria themselves. However, metal adsorption to bacteria is also reversible, suggesting that mobility within porous medium will depend on the interplay between adsorption-desorption kinetics and thermodynamic driving forces for adsorption. Till now there has been no systematic investigation of these important interactions. This study investigates the thermodynamic and kinetic controls of cotransport of Pantoea agglomerans cells and Zn in quartz and iron-oxide coated sand (IOCS) packed columns. Batch kinetic studies show that significant Zn sorption on IOCS takes place within two hours. Adsorption onto P. agglomerans surfaces reaches equilibrium within 30 min. Experiments in flow through quartz sand systems demonstrate that bacteria have negligible effect on zinc mobility, regardless of ionic strength and pH conditions. Zinc transport exhibits significant retardation in IOCS columns at high pH in the absence of cells. Yet, when mobile bacteria (non attached) are passed through simultaneously with zinc, no facilitated transport is observed. Adsorption onto cells becomes significant and plays a role in mobile metal speciation only once the IOCS is saturated with zinc. This suggests that IOCS exhibits stronger affinity for Zn than cell surfaces. However, when bacteria and Zn are preassociated on entering the column, zinc transport is initially facilitated. Subsequently, zinc partly desorbs from the cells and redistributes onto the IOCS as a result of the higher thermodynamic affinity for IOCS.

Enhanced resistance to nanoparticle toxicity is conferred by overproduction of extracellular polymeric substances.

The increasing production and use of engineered nanoparticles, coupled with their demonstrated toxicity to different organisms, demands the development of a systematic understanding of how nanoparticle toxicity depends on important environmental parameters as well as surface properties of both cells and nanomaterials. We demonstrate that production of the extracellular polymeric substance (EPS), colanic acid by engineered Escherichia coli protects the bacteria against silver nanoparticle toxicity. Moreover, exogenous addition of EPS to a control strain results in an increase in cell viability, as does the addition of commercial EPS polymer analogue xanthan. Furthermore, we have found that an EPS producing strain of Sinorhizobium meliloti shows higher survival upon exposure to silver nanoparticles than the parent strain. Transmission electron microscopy (TEM) observations showed that EPS traps the nanoparticles outside the cells and reduces the exposed surface area of cells to incoming nanoparticles by inducing cell aggregation. Nanoparticle size characterization in the presence of EPS and xanthan indicated a marked tendency towards aggregation. Both are likely effective mechanisms for reducing nanoparticle toxicity in the natural environment.

Inhibition of Sporosarcina pasteurii under anoxic conditions: implications for subsurface carbonate precipitation and remediation via ureolysis.

The use of Sporosarcina pasteurii to precipitate calcium carbonate in the anoxic subsurface via ureolysis has been proposed for reducing porosity and sealing fractures in rocks. Here we show that S. pasteurii is unable to grow anaerobically and that the ureolytic activity previously shown under anoxic conditions is a consequence of the urease enzyme already present in the cells of the aerobically grown inoculum. The implications are discussed, suggesting that de novo synthesis of urease under anoxic conditions is not possible and that ureolysis may decline over time without repeated injection of S. pasteurii as the urease enzyme degrades and/or becomes inhibited. Augmentation with a different ureolytic species that is able to grow anaerobically or stimulation of natural communities may be preferable for carbonate precipitation over the long term.

A lead isotopic study of the human bioaccessibility of lead in urban soils from Glasgow, Scotland.

The human bioaccessibility of lead (Pb) in Pb-contaminated soils from the Glasgow area was determined by the Unified Bioaccessibility Research Group of Europe (BARGE) Method (UBM), an in vitro physiologically based extraction scheme that mimics the chemical environment of the human gastrointestinal system and contains both stomach and intestine compartments. For 27 soils ranging in total Pb concentration from 126 to 2160 mg kg(-1) (median 539 mg kg(-1)), bioaccessibility as determined by the 'stomach' simulation (pH ~1.5) was 46-1580 mg kg(-1), equivalent to 23-77% (mean 52%) of soil total Pb concentration. The corresponding bioaccessibility data for the 'stomach+intestine' simulation (pH ~6.3) were 6-623 mg kg(-1) and 2-42% (mean 22%) of soil Pb concentration. The soil (206)Pb/(207)Pb ratios ranged from 1.057 to 1.175. Three-isotope plots of (208)Pb/(206)Pb against (206)Pb/(207)Pb demonstrated that (206)Pb/(207)Pb ratios were intermediate between values for source end-member extremes of imported Australian Pb ore (1.04)--used in the manufacture of alkyl Pb compounds (1.06-1.10) formerly added to petrol--and indigenous Pb ores/coal (1.17-1.19). The (206)Pb/(207)Pb ratios of the UBM 'stomach' extracts were similar (<0.01 difference) to those of the soil for 26 of the 27 samples (r=0.993, p<0.001) and lower in 24 of them. A slight preference for lower (206)Pb/(207)Pb ratio was discernible in the UBM. However, the source of Pb appeared to be less important in determining the extent of UBM-bioaccessible Pb than the overall soil total Pb concentration and the soil phases with which the Pb was associated. The significant phases identified in a subset of samples were carbonates, manganese oxides, iron-aluminium oxyhydroxides and clays.

Kinetics of bacterial potentiometric titrations: the effect of equilibration time on buffering capacity of Pantoea agglomerans suspensions.

Several recent studies have made use of continuous acid-base titration data to describe the surface chemistry of bacterial cells as a basis for accurately modelling metal adsorption to bacteria and other biomaterials of potential industrial importance. These studies do not share a common protocol; rather they titrate in different pH ranges and they use different stability criteria to define equilibration time during titration. In the present study we investigate the kinetics of bacterial titrations and test the effect they have on the derivation of functional group concentrations and acidity constants. We titrated suspensions of Pantoea agglomerans by varying the equilibration time between successive titrant additions until stability of 0.1 or 0.001 mV s(-1) was attained. We show that under longer equilibration times, titration results are less reproducible and suspensions exhibit marginally higher buffering. Fluorescence images suggest that cell lysis is not responsible for these effects. Rather, high DOC values and titration reversibility hysterisis after long equilibration times suggest that variability in buffering is due to the presence of bacterial exudates, as demonstrated by titrating supernatants separated from suspensions of different equilibration times. It is recommended that an optimal equilibration time is always determined with variable stability control and preliminary reversibility titration experiments.