ENVIRONMENTAL BIOTECHNOLOGY

Enhancement of the antimicrobial performance of biocidalformulations used for the preservation of whitemineral dispersions

Nicola Di Maiuta & Patrick Schwarzentruber &

Crawford S. Dow

Received: 30 June 2010 /Revised: 3 September 2010 /Accepted: 7 September 2010 /Published online: 28 September 2010# Springer-Verlag 2010

Abstract Biocides play an important role in the preserva-tion of white mineral dispersions (WMD). Due to theoccurrence of biocide-resistant bacteria and technicallimitations in the use of biocides, new preservationstrategies are required—like the enhancement of biocidesby non-biocidal compounds. The aim of this study was toevaluate the biocide enhancement performance of lithiumagainst various biocide-resistant bacteria in WMD. Subse-quently, the minimal enhancing concentration (MEC) oflithium and the bioavailability of lithium in respect to themode of introduction into WMD were investigated. Theantimicrobial performance of biocidal formulations com-prising isothiazolinones and formaldehyde releasers orisothiazolinones and glutaraldehyde has been evaluatedagainst the related resistant bacterial spectrum in thepresence of lithium. The MEC of lithium ranged from1,350 to 1,500 ppm (based on the liquid phase weight of aWMD with 75% solids) for formaldehyde releasers andglutaraldehyde-based biocidal formulations, respectively.The biocide enhancing property of lithium was independentof whether lithium was introduced into WMD via a lithium-neutralised dispersant, added during the calcium carbonategrinding step, or dosed into the final product. Lithium is anon-biocidal compound which has been discovered to be apotent and universal biocide enhancer. Lithium boosts thebiocidal activity of various biocides and provides a noveltechnique to overcome biocide resistance in WMD. Such a

biocide enhancer represents a breakthrough that offers apotential tool to revolutionise the consumption of biocidalagents in the WMD producing industry.

Keywords Biocide . Enhancement . Resistance . Bacteria .

Lithium . Calcium carbonate slurry .White mineraldispersion

Introduction

White mineral dispersions (WMD; ground and precipitatedcalcium carbonate, modified calcium carbonate, clay andtalc) are water-based mineral dispersions used as filler andpigment in the paper and paint industries (Rohleder andHuwald 2001). Although the alkaline pH of WMD rangesfrom 8 to 10, and the water content from 25% to 80% (w/w),the presence of various salts and the available oxygen aresufficient to promote an adequate microbial propagationenvironment. Bacterial contamination of WMD often leadsto acidification of the product. A decrease in pH is usuallyassociated with an increase in viscosity and consequentlyinvolves alteration of the rheological parameters such asfluidity and pumpability, which are required to ensuretrouble-free handling to the final user. Discolouration anddevelopment of unpleasant odours can occur under certainconditions when oxygenation of the WMD product stopsand aerobic growth is replaced by anaerobic growth.Therefore, biocides play an important role in the preserva-tion of WMD in order to maintain high hygiene and qualitystandards, such as brightness, rheologic parameters as wellas odour neutrality.

Due to the occurrence of biocide-resistant bacteria inWMD, as well as technical limitations on the use ofbiocides (e.g. acidic biocide formulations), there is increas-

N. Di Maiuta (*) : P. SchwarzentruberOmya Development AG, R&D Microbiology,4665 Oftringen, Switzerlande-mail: nicola.dimaiuta@omya.com

N. Di Maiuta :C. S. DowDepartment of Biological Sciences, University of Warwick,Coventry CV4 7AL, UK

Appl Microbiol Biotechnol (2011) 89:429–439DOI 10.1007/s00253-010-2884-9

ing demand for new preservation strategies to maintain thehigh quality required of WMD in respect of brightness,rheological parameters and odour neutrality (Di Maiuta etal. 2009). A lack of biocidal activity having beenrecognised, the only available options are to increase thebiocide dosage or to substitute the antimicrobial compoundcurrently in-use with another one (Schwarzentruber 2003;Schwarzentruber and Gane 2005). Apart from the economicand ecological impact caused by increased application ofbiocide, in this context, it is essential to highlight the factthat there exist only a limited number of antimicrobials thatare suited to preserving WMD. This is due to the morerigorous regulatory situation created by the BPD (BiocidalProducts Directive 98/8/EC), as well as the incompatibilityof certain antimicrobials with the chemical properties ofWMD. These circumstances have led to the shelving ofdevelopment and registration of new biocidal substances,owing to the laborious and expensive research required.(Kähkönen and Nordström 2008) in addition, some biocidalcompounds used for the preservation of calcium carbonateslurries, such as formaldehyde releasers, are under pressuredue to their possible classification as carcinogenics, whilesome EU member states have reduced their occupationalexposure limits. The few remaining actives such as glutar-aldehyde, isothiazolinones (CMIT/MIT/BIT), 2-bromo-2-nitropropane-1,3-diol (BNPD) or 2,2-dibromo-3-nitrilopro-pionamide (DBNPA) are less stable, while the acidiccharacter of some compounds constitutes a technical hurdlefor calcium carbonate slurries which are rather alkaline.

Of the strategies for overcoming biocide resistance andto keep within technical limitations on the application ofbiocides, one approach is to use combinations of antimi-crobial compounds. Combinations of biocides have beendescribed to exhibit a synergistic effect in that theeffectiveness of the combined antimicrobials is greater thanthe sum of the individual compounds (Lambert et al. 2003;Botelho 2000; Denyer et al. 1985, 1986; Acar 2000).However, the synergy is an effect brought about bycombining reduced concentrations of different antimicro-bials, rather than combining the in-use biocides with non-antimicrobial compounds. The term ‘enhancement’ istherefore more appropriate to describe an increase of theantimicrobial activity of a biocide by means of a non-antimicrobial compound (Hodges and Hanlon 1991).Previous studies have reported the enhancement of biocidesby non-antimicrobial compounds such as EDTA (Vaara1992; Ayres et al. 1999; Alakomi et al. 2006) andpolyethylenimine (Helander et al. 1997; Alakomi et al.2006; Khalil et al. 2008). Generally, the enhancementactivity has been suggested as originating from destabilisa-tion of the bacterial cell membrane of Gram-negativebacteria, thus increasing its permeability to the antimicro-bials. This effect has been reported in connection with a

vast assortment of other compounds such as polycations,lactoferrin, transferrin and citric acid (Hancock and Wong1984). Additionally, interference with the quorum sensingsystem (Vestby et al. 2009) or the substrate as a promoter ofthe uptake of antimicrobials has already been described(Maillard and Russell 2001; Denyer and Maillard 2002).Metals such as silver (Silvestry-Rodriguez et al. 2007),mercury (Morrier et al. 1998), copper (Sondossi 1990) andcaesium (Avery 1995) have also been noted to exhibitbiocide potential or even toxicity towards bacteria. Ingeneral, heavy metal ions are more toxic than the otherclasses of metal ions (Harrison et al. 2005).

Lithium has been determined as exhibiting antiviralactivity (Amsterdam et al. 1990), as well as antimicrobialactivity against Gram-negative bacteria (Eisenberg et al.1991), Gram-positive bacteria and synergistic activity whencombined with antibiotics (Lieb 2002, 2004). In addition,even though the molecular mechanism of electropermeabi-lisation is not fully understood, lithium is routinely used toperform lithium-cation induced electrotransformation ofbacteria. This effect is believed to be related to a transientincrease in permeability of the cell membrane (Papagianniet al. 2007; Ramon and Fonzi 2009).

Since lithium is easy to introduce into calcium carbonateslurry via dispersant neutralisation, the main objective ofthis study was to evaluate the enhancement performance oflithium to the antimicrobial activity of the biocidalformulations in-use in calcium carbonate slurries againstinherent biocide-resistant bacteria. The effects of concen-tration and the mode of introduction of lithium into calciumcarbonate slurry were determined. In addition, the inherentactivity and the mechanism of enhancement of lithium wereinvestigated.

Materials and methods

Chemicals and biocide dosage

A commercial biocidal formulation of (ethylenedioxy)dimethanol (EDDM, CAS No. 3586-55-8), 5-chlor-2-methyl-2H-isothiazolin-3-one (CMIT, CAS No. 26172-55-4)and N-methyl-isothiazolin-3-one (MIT, CAS No. 2682-20-4)was obtained from Dow Microbial Control (Horgen,Switzerland). A commercial biocidal formulation of glutar-aldehyde (GDA, CAS No. 111-30-8), CMIT and MIT wasobtained from Lanxess Deutschland GmbH (Leverkusen,Germany). Unless otherwise stated, all other chemicals andthe culture media were purchased from Sigma–Aldrich(Buchs, Switzerland) or Becton Dickinson (Allschwil,Switzerland), respectively.

In liquid cultures, commercial biocide formulations weredosed in parts per million (w/w) of the commodity based on

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the total weight of the solution. In contrast, the biocideamount dosed in calcium carbonate slurry is reported inparts per million (w/w) of the commodity relative to theweight of the aqueous phase of a WMD with a solid contentof 75% (w/w).

Manufacture of calcium carbonate slurry

Calcium carbonate slurry is typically prepared by wetgrinding marble in the presence of 0.2% to 1.3% (w/w) of aradically polymerised polyacrylic acid wherein the carbox-ylic acid groups are neutralised by alkali metals. In thisstudy, Hydrocarb 90–75% (HC90, 75% solids content,pH 9 to 9.5, and particle size 90%<2 μm) was used. Sterileslurry was prepared by sterilisation at 121 °C for 15 min inan autoclave. Lithium was introduced into the calciumcarbonate slurries via the dispersant (fully or partiallyneutralised with lithium) or during the wet grinding ofmarble in the form of lithium carbonate, as well as added tothe final slurry product by means of a lithium carbonatesolution. The nomenclature and details of the calciumcarbonate slurries investigated in this study are summarisedin Table 1.

Identification of microorganisms in WMD and cultureconditions

The biocide-resistant cultures used in this study have beendeposited in the Culture Collection of Switzerland (CCOS)repository. The biocide-resistant calcium carbonate slurrycultures, GDA/CMIT/MIT-resistant (rGCM; CCOS 29) andEDDM/CMIT/MIT-resistant (rECM; CCOS 30), wereobtained from product storage tanks located at WMDmanufacturing plants that used the related biocide topreserve the WMD. The in-use concentrations of GDA/CMIT/MIT formulation and EDDM/CMIT/MIT formulationwere 1,350 and 750 ppm, respectively. The formaldehyde-resistant WMD cultures of Pseudomonas putida (CCOS 31)and facultative methylotrophic Methylobacterium extorquens(CCOS 32) were described previously (Di Maiuta et al.

2009). Biocide-resistant colonies isolated from biocide-supplemented calcium carbonate slurry were phylogeneticallyidentified by partially sequencing the 16S rRNA gene (DiMaiuta et al. 2009). WMD bacteria cultures utilised for thedetermination of the antimicrobial activity of biocides arebatch culture which have been standardised by incubationtime (statically for 48 h at 30 °C and TVC >105 cfuml−1).Fifty grams of fresh sterile Hydrocarb 90–75%, containingthe corresponding concentration of biocide if required, wasinoculated with 1 ml of the preceding slurry culture. After48 h of incubation, bacterial growth was confirmed byplating on PCA plates a 1:10 dilution of the slurry inphosphate buffered saline. The slurry cultures provided thebasis for inoculation of the challenge testing assays incalcium carbonate slurries.

Challenge testing

Fifty-gram aliquots of sterilised calcium carbonate slurrywere each supplemented with different concentrations ofbiocide formulation. An aliquot containing no biocide wasincluded to ensure that the used WMD cultures grow in theunpreserved product. The samples were then incubated for3 days at 30 °C. Subsequently, the samples were inoculatedwith 1 ml of contaminated WMD culture as describedabove. After 24 h to 3 days of incubation at 30 °C, the totalviable count was determined according to the plate countmethod. Samples that did not show any counts (<100 cfuml−1) were further inoculated with 1 ml of the same WMDculture as above. No more than three inoculations wereperformed. Cell counts greater than 104 cfuml−1 wereevaluated qualitatively by estimating the order of magni-tude (105, 106 or higher). All analyses have been repeatedtwo times with high reproducibility.

Lithium ion analysis

Slurry liquid phase was extracted by pressure filtration(Fann Instruments filter press series 300, special hardenedfilter paper 3.500, retention 2–5 μm, 6 bar) and passed

Table 1 Details of the calcium carbonate slurry products used in this study

Product ID Dispersant neutralisation (%) Lithium contenta (ppm) Lithium measuredb (ppm) pH Remarks

HC90-Li 100 Li 1,580 407 Lithium via dispersant

HC90-MgNa5050 50:50 Mg:Na 0 0 9.2 Standard HC 90 slurry

HC90-MgLi5050 50:50 Mg:Li 920 ND 9.4 Lithium via dispersant

HC90-MgLi7525 75:25 Mg:Li 660 ND 9.4 Lithium via dispersant

HC90-gLi 50:50 Mg:Na 1,500 711 9.6 Ground with Li2CO3

ND not determineda Theoretical valuebMeasured by IC in the water phase

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through a Sartorius (Dietikon, Switzerland) 0.2-μm poresize syringe filter (Minisart RC) prior to measurement oflithium. Lithium concentration was determined by ionchromatography using a Metrohm (Zofingen, Switzerland)882 Compact IC plus system with autosampler. Twentymillilitres of filtrate was injected into a Metrosep A Supp 5(150×4.0 mm) column. The eluent (flow rate 0.7 mlmin−1)composition was NaHCO3 (1 mmoll−1)–Na2CO3 (3.2 mmoll−1) 1:1v/v. The analysis was performed at room temperature.

The total lithium concentration was determined bymeans of inductively coupled plasma optical emissionspectrometry (ICP–OES). Firstly, the water of thecalcium carbonate slurry samples was removed bydrying the sample at 120 °C. Eight millilitres of 69%HNO3 was then added to 2 g of dried sample and boiledfor 5 min to carry out digestion. Finally, the boiled samplewas cooled to 20 °C and adjusted to 100 ml with ultrapurewater (Milli-Q® integral water purification system). TheOPTIMA 3200 XL ICP–OES system from Perkin Elmer(Schwerzenbach, Switzerland) was used. Lithium ionswere detected in the emission line at wavelengths of610.35 and 670.76 nm.

Leakage of cellular constituents caused by lithium

Pseudomonas mendocina cells isolated from the EDDM/CMIT/MIT-resistant culture were used to investigate theeffect of lithium on bacterial cells. Bacteria were grown intryptic soy broth at 30 °C and 160 rpm on an orbital shakerovernight (15±1 h). Cells were harvested by centrifugation(5,000 rcf for 10 min), washed twice with 10 mmoll−1

HEPES buffer pH 7.4 (Denyer et al. 1986) and resuspendedin HEPES buffer to achieve a cell density of 107 to108 cellsml−1. The HEPES buffer–cell suspension was thensupplemented with biocide, lithium or a combinationthereof and analysed at 0, 30 and 60 min after therespective treatments. Two biological replicates and twotechnical replicates of all samples were analysed.

The absorbance measurements at 600 and at 260 nm wereperformed using a Vaudaux-Eppendorf (Basel, Switzerland)Bio-Photometer. Prior to A260 analysis and determination ofthe potassium ion concentration, the suspension was passedtrough a 0.2-μm pore size syringe filter to remove the cells.The potassium ion concentration was measured by means ofICP–OES. Potassium ions were detected in the emission lineat a wavelength 766.47 nm as described above.

Membrane permeabilisation and depolarisation assay

The NPN (1-N-phenylnaphthylamine) uptake assay wasperformed to assess membrane permeabilisation as describedby Helander and Mattila-Sandholm (2000). Fluorescencewas detected using a Synergy™ HT microplate reader

(BioTek Instruments Inc., Winooski, VT, USA). Wells wereread from the bottom with a sensitivity value of 70, andfilter settings were 360/40 nm for the excitation and 420/50 nm for the emission. P. mendocina cells were preparedas described for the leakage experiments and resuspendedin 5 mmoll−1 HEPES pH 7.2. The NPN stock solution(0.5 mmoll−1) was prepared in acetone. For each substance,16 replicates (wells) in a 96-microtitre plate were analysed(two biological replicates and eight technical replicateswere analysed). Fifty microlitres NPN (40 μmoll−1, dilutedin 5 mmoll−1 HEPES pH 7.2) and 25 μl of each substance(in HEPES buffer; 8× higher in order to achieve the desiredfinal concentration in the well) were adjusted to a totalvolume of 100 μl with HEPES buffer if required. Justbefore measurement, 100 μl of bacterial suspension wasadded to the wells, the plate briefly shaken for 15 s andfluorescence values recorded within 5 min. Controls includedbuffer alone (200 μl), buffer (100 μl) and bacterial suspen-sion, buffer (150μl) and NPN (50μl) as well as buffer (50μl),NPN (50 μl) and bacterial suspension (100 μl). The relativefluorescence unit value (rfu) was calculated as follows: rfu=(cells+test substance+NPN)−(cells+NPN). The NPN factorwas calculated as the ratio of the rfu value of the substance(s)to the rfu value of the NPN supplemented buffer background[(buffer+NPN)−(buffer)]. Control experiments includedtreatment of the cells with saline solution (0.9% NaCl) orETDA. Statistically significant changes of the NPN uptakefactor were determined by the Student's t test (unpaired, equalvariance) based on the untreated sample (buffer only).

The CellFacts II® analyzer from CellFacts Instruments(Coventry, UK) was used to assess membrane depolarisation.Two hundred fifty grammes sterilised calcium carbonate slurrywas inoculated with 5 ml of rECM culture, and afterincubation for 48 h at 30 °C, 50 g aliquots were eachsupplemented with different amounts of lithium. Lithium wassupplemented in the form of lithium carbonate. Membranepotential of the cells was determined 30 min after the additionof lithium to the slurry cultures as described by DiMaiuta et al.(2009) with minor modifications. To stain the cells, 5 μl of100 μM 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5),Invitrogen) was added into the tube, mixed well andincubated in darkness for 15 min. The CellFacts II®instrument was operated according to the manufacturer'sinstructions. Two biological replicates and two technicalreplicates were analysed.

Results

Characterisation of biocide-resistant bacteria in WMD

Biocide-resistant colonies isolated from biocide-supplementedcalcium carbonate slurry were identified by sequencing

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approximately 800 bp of the 16S rRNA gene. Sequenceanalysis of the isolated strains revealed that both resistantcultures rGCM and rECM contained bacteria belonging to thegenusPseudomonas ( Table 2). The closest relatives based onthe similarity of the 16S rRNA gene sequence were thespecies Pseudomonas pseudoalcaligenes, P. mendocina andPseudomonas alcaliphila. However, by means of the 16SrRNA gene, the resolution between the Pseudomonas spp. islimited (Mulet et al. 2010).

Resistance was analysed by means of the challenge test.The MIC of the biocides against the corresponding resistantbacteria were determined as the concentration preventingbacterial growth detection over three cycles of inoculation.The resistance and the susceptibility level of the biocide-resistant bacteria were compared to the MIC of the biocide-susceptible WMD bacterial population H90-i found inslurry produced under laboratory conditions in small scale(Pseudomonas sp.). In comparison to the H90-i culture, theincrease in biocide concentrations used to preserve calciumcarbonate slurry over three bacteria challenges withbiocide-resistant bacteria was 20- and ninefold higher forthe biocidal formulations GDA/CMIT/MIT and EDDM/CMIT/MIT, respectively. These MIC values clearly showedthat the WMD samples obtained from the biocide-usingproduction plants were contaminated with bacteria thatwere adapted or even resistant to the applied biocideformulation.

Lithium content of the investigated calcium carbonateslurries

The lithium concentrations summarised in Table 1 showthat the determined lithium concentration of slurry deviatedsignificantly from the theoretical calculated value based onthe dosed lithium-neutralised dispersant or lithium carbon-ate amount, respectively. The lithium concentration wasinitially determined by means of ion chromatography in theaqueous phase of the calcium carbonate. These datasuggested that the lithium introduced into the calciumcarbonate slurry via dispersant was not completely dis-

solved in the water phase and not completely dissociatedfrom the polyacrylate dispersant. Similarly, lithium intro-duced into the calcium carbonate slurry in the form oflithium carbonate reacted with the polyacrylates and waspartially sequestered to neutralise free carboxyl groups ofthe polyacrylate polymer. These observations were corrob-orated by the fact that using ICP–OES to determine thetotal lithium content revealed a concentration of 1,499 ppmlithium for the H90-Li slurry manufactured with fullylithium-neutralised dispersant.

Addition of lithium in WMD via neutralised dispersant

The enhancement efficacy of lithium to EDDM biocide wasinvestigated in various calcium carbonate slurries wherelithium was introduced into the product via lithium-neutralised dispersant ( Table 3). After a single challengewith the formaldehyde-resistant WMD cultures of P. putidaor facultative methylotrophic M. extorquens, significantgrowth of both bacterial species was observed in allcalcium carbonate slurry products without biocide. How-ever, product HC90-Li dispersed with lithium-neutraliseddispersant showed inhibition of both formaldehyde-resistantmicroorganisms when 1,500 ppm EDDM biocide had beenadded to the slurry. On the other hand, the HC90 slurriesmanufactured by means of dispersant neutralised with only25% or 50% all showed considerable bacterial contamina-tion. The biocide-enhanced performance to EDDM of aHC90-Li dilution series in conventional lithium-free HC90-MgNa5050 was also examined ( Table 4). These resultsindicated that a 1:2 dilution of HC90-Li with conventionalHC90 reduced the lithium content significantly. In thediluted slurries, a lack of lithium-derived biocide-enhancedefficacy against both M. extorquens and Pseudomonas sp.formaldehyde-resistant bacteria was determined.

Addition of lithium into WMD via lithium carbonate

The biocide-enhancing performance of lithium introducedinto WMD in the form of lithium carbonate was determined

Table 2 Calcium carbonate slurry isolated species from the biocide-resistant culture identified on the basis of 16S rRNA gene sequencealignment, with use of BlastN and RDPII databases

Taxonomic affiliation Closest relativea Accession No.b Maximum identityc Length (bp)

Pseudomonas Pseudomonas pseudoalcaligenes AB109888 99 756

Pseudomonas mendocina AF232713 99 778

Pseudomonas alcaliphila AB030583 99 925

a Closest relative from comparison with BlastN and RDPII databasesb Accession number of the closest relative entrycMaximum identity for the covered sequence length

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by comparing the addition of lithium carbonate during themineral grinding step with the dosing of lithium carbonatedirectly into the final product. The calcium carbonate slurryHC90-gLi ground in the presence of lithium carbonate(1,500 ppm lithium) showed excellent enhancing properties( Fig. 1). Independent of which biocide formulation wasused, a concentration of 1,500 ppm lithium was sufficientto preserve calcium carbonate slurry over three bacterialchallenges with the related biocide-resistant culture.

The minimal enhancing concentration (MEC) of lithiumwas elucidated for both biocidal formulations against theirrespective biocide-resistant microorganisms by dosingvarious concentrations of lithium carbonate into regularHC90-MgNa5050 calcium carbonate slurry ( Figs. 2 and 3).The MEC of lithium for the biocide formulation EDDM/CMIT/MIT and GDA/CMIT/MIT over three challengeswith biocide-resistant bacteria were determined to be 1,050and 1,500 ppm, respectively. No significant differences intotal viable counts were observed in either biocide-free/lithium-containing calcium carbonate slurry samples or viceversa inoculated with the biocide-resistant bacteria culture.

The challenge testing results clearly show that thebiocide enhancing property of lithium is dose-dependent,and that the slurry culture rECM was more susceptible tothe enhancing effect of lithium. A closer investigation ofthe enhancing concentration of lithium for a single bacterialchallenge revealed that complete eradication of the resistantbacteria was achieved at a dose of between 750 and1,050 ppm.

As a consequence of lithium showing an antimicrobialenhancing performance to different biocides against the

relative resistant slurry bacteria, it was assumed that othermonovalent cations such as potassium or sodium might alsobe potential candidates to enhance the performance of thebiocide. Sodium did not come into consideration since it isbeing used for neutralisation of the dispersant whereaspotassium in the equivalent concentration to lithium(1,500 ppm) did not show any enhancement of theantimicrobial performance of the biocides EDDM/CMIT/MIT and GDA/CMIT/MIT against the relative resistantslurry cultures (data not shown).

Effect of lithium on P. mendocina cells isolated from WMD

P. mendocina cells isolated from the EDDM/CMIT/MIT-resistant culture were used to investigate the influence oflithium on the bacterial cells on its own and in the presenceof biocide formulation EDDM/CMIT/MIT. The leakage ofcytoplasmic constituents absorbing at 260 nm (such asnucleotides, nucleosides and aromatic amino acids) alongwith potassium was recorded over a time period of 60 minafter exposing the cells to lithium, biocide or combinationsthereof ( Fig. 4a, b). Changes in cell morphology (lysis orswelling) were followed by monitoring the absorbance at600 nm ( Fig. 4c).

Firstly, in bacterial cells exposed to 750 ppm of thebiocide mixture EDDM/CMIT/MIT only, or in combinationwith 1,500 ppm lithium, a significant leakage of cellularconstituents absorbing at 260 nm was observed. Thirtyminutes after the treatment, the absorbance (260 nm)increased to an average level of 0.28 units, which isequivalent to a secretion of 13.5 μgml−1 of dsDNA

Product ID Total viable count (cfuml−1) as mean (standard deviation)

M. extorquens Pseudomonas sp.

EDDM (ppm) 0 1,500 0 1,500

HC90-MgNa5050 >106 3.2×104 (4×103) >106 1.6×104 (3.3×103)

HC90-Li >106 <102 >106 <102

HC90-MgLi5050 >106 4.4×104 (9.6×103) >106 8.0×104 (7.1×103)

HC90-MgLi7525 >106 2.4×104 (3.7×103) >106 1.6×104 (6.8×103)

Table 3 Enhancement of thebiocidal activity of EDDM byvarious concentrations oflithium introduced into WMDvia dispersant

Product Total viable count (cfuml−1) mean (standard deviation)

M. extorquens Pseudomonas sp.

HC90-MgNa5050 >106 >106

HC90-Li <102 <102

Dilution ratiosa

1:2 >106 3.2×104 (1.4×104)

1:4 >106 5.0×104 (7.4×103)

1:8 >106 5.0×104 (1.4×104)

Table 4 Enhancement perfor-mance of lithium in serialdilutions of HC90-Li to1,500 ppm EDDM biocideagainst formaldehyde-resistantWMD bacteria

a HC90-Li:HC90-MgNa5050

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(1.0 A260 unit=50 μgml−1). In contrast, the sameconcentration of lithium on its own did not show any effecton the leakage of cellular constituents determined at260 nm. On the other hand, 1,500 ppm of lithium induceda rapid outflow of potassium ions either on its own or incombination with 750 ppm EDDM/CMIT/MIT biocide,ranging from 14 to 18 ppm. Morphological effectsmonitored at 600 nm were observed in bacterial cellsexposed to lithium or lithium combined with the biocide.An increase of absorbance at 600 nm probably indicatedthat the treatment with lithium resulted in a cellularswelling or promoted cytokinesis. Secondly, the permeabi-lisation of P. mendocina treated with lithium, EDDM/CMIT/MIT biocide blend or a mixture was assessedutilising the NPN uptake assay. The NPN uptake factorsare shown in Fig. 5. Lithium on its own (1,500 ppm) wassignificantly (p<0.01) permeabilising P. mendocina cells asdemonstrated by the enhanced uptake of NPN. Similarly,lithium in combination with the biocide mixture EDDM/CMIT/MIT (750 ppm) showed a reduced but still signifi-cantly increased uptake of NPN compared to the untreatedcontrol (p<0.01). Control experiments with EDTA(180 ppm) instead of biocide/lithium showed a significantincreased uptake of NPN too. In contrast, cells treated only

with 750 ppm of the biocide EDDM/CMIT/MIT mixturedid not show significant uptake of NPN. Similar resultswere obtained by replacing the biocide/lithium volume withsaline solution (0.9% w/w NaCl), indicating that thepermeabilisation effect is fully attributable to the effect oflithium on the bacterial membrane. Finally, using thefluorescent dye DiSC3(5), a significant depolarisation ofthe bacterial membrane in the presence of lithium wasobserved (Fig. 6).

Discussion

The development of biocide resistance and the incidence ofmultiple biocide-resistant bacteria in calcium carbonateslurries is a primary factor determining future preservationstrategies for WMD. For this reason, WMD preservationresearch activities have been increased on novel andinnovative preservation approaches. Synergistic combina-tions of biocidal actives such as aldehyde-based or aldehyde-releasing compounds with isothiazolinones, BNPD orDBNPA have been used for many years to preserve WMD(Schwarzentruber 2003; Schwarzentruber and Gane 2005).Nevertheless, WMD-contaminating bacteria resistant toseveral combinations of biocidal actives have been discov-ered. The estimation of synergy between combinations ofantimicrobials has been described in detail as the sum of thefractional inhibitory concentration assessed by means of thecheckerboard titration principle (Berenbaum 1978). How-ever, using fractional inhibitory concentrations to investi-gate synergistic effects of antimicrobials assumes a lineardose response, and it has been suggested that the synergismoccasionally arises from the combination of antimicrobialswith different dose-responses, rather than from theirsynergistic performance (Lambert and Lambert 2003;Lambert et al. 2004, 2003). In the case of WMD, amodified definition of the terms biocide synergy andenhancement have been introduced: (1) a synergisticbiocidal formulation is considered to be a blend of two ormore antimicrobial actives to preserve WMD, and (2) a

Fig. 1 Enhancement of EDDM/CMIT/MIT and GDA/CMIT/MITbiocide formulations in HC90-gLi calcium carbonate slurry ground inthe presence of lithium carbonate. Calcium carbonate samples wereinoculated with the resistant bacteria rECM and rGCM, respectively

Fig. 2 Biocide enhancementperformance of lithium againstrECM WMD culture in thepresence of 750 ppm EDDM/CMIT/MIT determined bymeans of the challenge test.For each sample, a three-cyclechallenge test was performed.Total viable count (TVC)detection limit was 102 cellsml−1

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biocide enhancer is a substance that is not noted to be abiocide and which at the applied concentration does notexhibit antimicrobial activity in WMD.

The results of this study clearly showed that lithium is auniversal enhancer of biocides used for the preservation ofWMD. WMD bacteria with resistance to the in-use biocideconcentration were susceptible to the biocide in the

presence of lithium ions. The major advantage is thatlithium can be introduced into WMD simply via dispersantneutralisation. No difference in the biocide enhancerproperties of lithium was observed whether it was intro-duced into the calcium carbonate slurry during the grindingprocedure or afterwards into the final product. In thiscontext, it is important to mention that for lithium, the

Fig. 3 Biocide enhancementperformance of lithium againstrGCM WMD culture in thepresence of 1,350 ppm GDA/CMIT/MIT determined bymeans of the challenge test.For each sample, a three-cyclechallenge test was performed.Total viable count (TVC)detection limit was 102 cellsml−1

a b

c

Fig. 4 Pseudomonas mendocina leakage of cytoplasmic componentsat 260 nm (a) and potassium (b) as well as absorbance at 600 nm (c)measured 30 and 60 min after exposure to the following combinations:no biocide/lithium control (black square), 750 ppm EDDM/CMIT/

MIT (white circle), 1,500 ppm lithium (white triangle) and 750 ppmEDDM/CMIT/MIT+1,500 ppm lithium (black circle). All concen-trations are given in parts per million based on the weight of thesolution. Values are presented as mean±standard deviation (n=4)

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fractional inhibitory concentration index (FICI) calculatedaccording to Berenbaum (1978) revealed a FICI value of0.8 for the EDDM/CMIT/MIT biocide and 0.7 for theGDA/CMIT/MIT biocide, respectively. Synergism of drugsor antimicrobials has been described as occurring when theFICI is <1 (Berenbaum 1978); nevertheless, that interpreta-tion was revised by the American Society of Microbiology,which has defined a synergy at FICI<0.5. Controversially,other authors have defined a partial synergy as 0.5>FICI<1(Botelho 2000). Therefore, in view of the fact that lithiumis not an antimicrobial, yet enhances the antimicrobialperformance of biocides in calcium carbonate slurries, it ismore appropriate to use the term ‘enhancement’ rather thansynergism.

Lithium has in fact been used since the 1950s to treatbipolar disorders in psychiatry (Cruceanu et al. 2009), andseveral clinical observations indicated that lithium mightposses an antimicrobial and antiviral performance both invitro and in vivo (Amsterdam et al. 1990; Lieb 2002, 2004,2007). There are a few studies that draw differingconclusions about the antimicrobial activity of lithium ionsagainst bacteria. A recent study found that the enzymealcohol dehydrogenase of M. extorquens was inhibited to28% by 5 mmoll−1 lithium (34.5 ppm; Koutsompogeras etal. 2006). Others authors reported a toxicity of lithiumtoward Escherichia coli at a concentration of 4,830 ppmand the arrest of cell division and differentiation ofprotozoa of the group Trypanosomatidae by 200 mmoll−1

LiCl (1,380 ppm Li+; Spiegel and Soares 1999). Someother authors have demonstrated that 100 mmoll−1 LiCl(690 ppm Li+) in the presence of various sole carbonsources inhibited the growth of Salmonella thyphimuriumand E. coli due to an accumulation of Li+ in the cellcytoplasm that might cause inhibition of the enzyme

pyruvate kinase (Niiya et al. 1980; Umeda et al. 1984),whereas others ruled out the enhancement of the antimi-crobial performance of fluoride by lithium (Eisenberg et al.1991).

Within the scope of this investigation, attempts weremade to hypothesise the enhancement mechanism oflithium in the presence of biocide. Experimental datashowed that an outflow of cellular constituents absorbingat 260 nm only took place in the presence of the biocideblend (EDDM/CMIT/MIT), suggesting that the release iscaused by interaction of the cell envelope with bothformaldehyde and isothiazolinones. In fact, neither formalde-hyde nor isothiazolinones are membrane-active biocides—thekind that disrupt the cell membrane and thus cause an outflowof cytoplasmic constituents (McDonnell and Russell 1999).However, the release of cell wall components absorbing at260 nm might be related to the initiation of cellular autolysiscaused by formaldehyde (Denyer 1995; Musterman andMorand 1977) and the rapid association of CMIT/MIT withthe cells (Diehl and Chapman 1999). On the other hand, anoutflow of potassium ions was observed only when lithiumwas present alone or in combination with the biocide(EDDM/CMIT/MIT). The greatest efflux was observed withthe combination of lithium and biocide. A leakage ofpotassium has been described as the first sign of an increasein membrane permeability and biocide-induced membranedamage (Lambert and Hammond 1973). Furthermore, thepresence of lithium leads to an increase of absorbance at600 nm, a swelling of the cells that is probably the result ofwater diffusing into the cell due to a rise in salinity. Previous

Fig. 5 NPN uptake factor obtained after exposing Pseudomonasmendocina cells to different treatments. The following concentrationswere used: 750 ppm EDDM/CMIT/MIT, 1,500 ppm lithium, 750 ppmEDDM/CMIT/MIT+1,500 ppm lithium and 1,500 ppm NaCl180 ppm EDTA. Values are presented as mean±standard deviation(n=16); **p<0.01 for Student's t test (unpaired, equal variance) basedon the untreated sample

Fig. 6 Influence of increasing lithium concentrations on the mem-brane potential of rECM WMD culture cells in standard calciumcarbonate slurry determined by means of the membrane potentialsensitive dye Disc3(5) using the CellFacts II® instrument. Lithium wasadded to the slurry culture, and fluorescence was determined after30 min. Values are presented as mean±standard deviation of twoindependent replicates

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studies described an increase of the absorbance at 600 nm tobe related to an alteration of the refractive index, the releaseof light scattering material or to swelling of the cells due tointracellular accumulation of biocide (Sheppard et al. 1997;Chun and Hancock 2000). Another possible explanation forthe slight increase in absorbance at 600 nm is the effect oflithium on bacterial cell division. This might result in thepromotion of the cytokinesis within the bacterial populationand thus to a shift of the average population size to smallercells, as well as to a change in the refractive index. In asimilar fashion, the data provided by the NPN uptake testdemonstrated that lithium significantly increased the perme-ability of the cell membrane to the hydrophobic NPN probe.

Based on the results, it can be assumed that the biocide-enhancing action of lithium is the consequence of interfer-ence with the Na+(Li+)/H+-antiporter systems, and in turn,the cation balance of the bacterial cells is compromised.While the cytoplasmic membrane is not permeable tocations without the involvement of membrane transportsystems, the Na+(Li+)/H+-antiporter exchanges a cytoplas-mic Na+ or Li+ for an external H+ (Inaba et al. 1994, 1997).In an alkaline environment, like that found in calciumcarbonate slurries, monovalent ions and especially Na+ ionsare key players in pH homeostasis, regulation of the Na+

content and cell volume (Padan et al. 2005; Hunte et al.2005). Moreover, these cationic antiporters are alsoinvolved in the cationic detoxification of the cells sincethe cytotoxicity of excess sodium and lithium increases asthe pH rises, and depends on the cytoplasmic K+

concentration (Padan et al. 2005; Hunte et al. 2005). Anincreased cationic load as a result of dosing lithium into thealkaline calcium carbonate slurry probably results in a toxicaccumulation of Na+ ions due to the competitive saturationof the antiporter system with Li+. The elevated extrusion ofLi+ ions consequently influences the transmembrane elec-trochemical gradient of protons or even the transmembranepH gradient. Other authors postulated that the efflux ofpotassium ions probably results from the dissipation of thetransmembrane electrochemical gradient of protons (Krolland Patchett 1991). In calcium carbonate slurry supple-mented with lithium, a depolarisation of the bacterial cellmembrane and hence the loss of membrane integrity wasobserved by means of the fluorescent dye DiSC3(5). As aconsequence, dissipation of the proton motive force leads tothe leakage of essential molecules (Friedrich et al. 1999).Another possible explanation is that either the formalde-hyde or the isothiazolinones deriving from the EDDM/CMIT/MIT biocide inhibited the antiporter system thuscausing an inhibitory growth effect because of the disabledextrusion of surplus cations. Finally, lithium and magnesiumare alike in some chemical and physical characteristics such astheir atomic and ionic radii; these similarities are known as adiagonal relationship. Chemical resemblances might lead to an

interaction between lithium and the magnesium ions anddestabilise the lipopolysaccharide structure of the cell mem-brane in a fashion similar to the metal chelator EDTA.

In calcium carbonate slurries, the interference of lithiumwith the cationic antiporter is probably also supported bythe increased pH observed after lithium carbonate isintroduced. However, an enhancement of the biocide dueto the alkaline stress caused by the pH shift in the presenceof lithium carbonate can be ruled out. Control experimentsusing potassium carbonate instead of lithium carbonate didnot show any biocide enhancement activity.

Summing up, experimental data provided evidence thatlithium enhances the biocidal activity of various biocidesand provides a novel technology to minimise biocideresistance in WMD. Additionally, lithium can be introducedinto calcium carbonate slurries via the dispersant efficientlyand without any additional dosage steps. This approachmay solve the emerging problems of bacterial resistance oradaptation by using an enhancer compound to assure theeffectiveness of the biocide at its action site. Such a biocideenhancer represents a breakthrough that offers a potentialtool to revolutionise the consumption of biocidal agents inthe WMD producing industry. Two final items of particularimportance are the specifications of enhancer compounds,which must be chemically compatible with WMD, and theeconomic and ecological sustainability of their deployment.

Acknowledgments We thank Daniel Oschwald, Michael Jäggi,Daniel Schild and Karin Fischer for their technical assistance withanalytical measurements. We also thank Jan Sinstadt for his criticalreading of the manuscript.

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