Abstract Lithium chloride, more specifically the lithi-um cation, has been implicated in interference in biologi-cal systems. In the case of Escherichia coli, interferenceinvolves the Na+(Li+)/H+ antiporter transport system.The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegradeboth methanol and acetone under aerobic conditions. Theresults obtained using unsteady state continuous flowculture techniques demonstrate a significant disruptiveeffect of LiCl on culture performance. In addition, a re-duction in the substrate-based biomass yield coefficient,which is a clear advantage as far as biotreatment processperformance is concerned, also occurs. The ultimate fateof the LiCl was not determined.

Introduction

Lithium compounds have various applications in the alu-minium, glass, ceramic and pharmaceutical industries.Lithium is a minor constituent of the earth’s crust with abackground concentration of 7–200 mg/kg dry matter asLi+ in the uncontaminated lithosphere and in the hydro-sphere, concentrations of 0.17 mg/dm3 and 1.1 µg/dm3

occur in seawater and fresh water, respectively (Jensenand Jørgensen 1984). Therefore, lithium is considered tohave low natural abundance and does not feature eitheras one of the four elements required in bulk or amongthe 26 elements required in either intermediate or tracequantities for microbial growth (Woods 1984). However,low concentrations of non-essential metals are generallyconsidered to exhibit toxic and/or inhibiting effects onmacro- and microorganisms, including bacteria. The

present concern is whether LiCl present in an industrialwastewater stream might have adverse effects on the per-formance of biotreatment processes during organic pol-lutant oxidation.

Early studies concerning the effects of inorganic salts,most commonly chlorides, on bacterial growth involvedsalt additions to complex growth media. Hotchkiss(1923) studied 22 different metal chlorides in this man-ner using Bacterium coli (Escherichia coli) with a viewto identifying both stimulating and inhibitory effects.The general trend was that growth stimulation occurredat lower salt concentrations and growth inhibition oc-curred at higher salt concentrations. In the specific caseof LiCl, stimulation was reported between 1.06 and5.3 g/dm3 LiCl, and complete inhibition at 31.4 g/dm3

LiCl. An extension to the study was carried out by Winslow and Haywood (1931). In contrast, they foundthat growth stimulation first occurred at 0.25 g/dm3 LiCl,but reached a maximum at 1.06 g/dm3 LiCl, and that inhibition occurred with 2.12 g/dm3 LiCl. In a study concerning the effect of salts on the endogenous respi-ration of Bacillus cereus, Ingram (1939) reported thatLiCl stimulated respiration at concentrations between0.42 and 4.2 g/dm3 LiCl. In a study with an osmo-philic yeast, Zygosaccharomyces major (Saccharomyces rouxii), Onishi (1957) reported that severe inhibitory effects were observed with LiCl. With 10.85 g/dm3 LiCl,the lag phase was increased about six-fold and the spe-cific growth rate was reduced around 4.5 times com-pared with cultures where no LiCl was present. In fact,21.7 g/dm3 LiCl completely inhibited yeast growth.

More recently a series of papers concerning the effects of Li+ on E. coli (Kayama and Kawasaki 1976;Kayama-Gonda and Kawasaki 1979; Tsuchiya et al.1984) have concerned themselves with the co-transportof proline and Li+, and in another study Umeda et al.(1984) showed that Li+ inhibited the growth of E. coliwhen glucose, galactose, fructose and glycerol were sup-plied as sole carbon energy substrates, but that when either lactate or a mixture of amino acids were suppliedas the carbon source, no inhibition occurred. It was

M. O’Brien · G. Hamer (✉ )Chemical Engineering Department, University College Dublin,Belfield, Dublin 4, Irelande-mail: chemical.eng@ucd.ieFax: +353-1-7161177

Present address:M. O’Brien, Jones Environmental Ireland, Kingswood Drive, City West Business Campus, Dublin 24, Ireland

Appl Microbiol Biotechnol (2001) 56:508–512DOI 10.1007/s002530100615

S H O RT C O N T R I B U T I O N

M. O’Brien · G. Hamer

The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures

Received: 23 October 2000 / Received revision: 11 December 2000 / Accepted: 15 December 2000 / Published online: 24 May 2001© Springer-Verlag 2001

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postulated that intracellular Li+ most probably inhibitsthe glycolytic pathway. Finally, Inaba et al. (1994)sought to explain Li+ toxicity in E. coli on the basis thatit results from a failure by two Na+(Li+)/H+ antiportersystems to extrude Li+ from the cells at higher extracel-lular Li+ concentrations. They showed that total inhibi-tion occurred at LiCl concentrations of 30.4 g/dm3 LiCl,but that at 26.0 g/dm3 LiCl, growth occurred as a resultof the effective operation of one antiporter system. Thesecond antiporter system only functioned at LiCl con-centrations below 8.68 g/dm3.

The present study involves the effect of LiCl on the aerobic biodegradation of methanol and acetone,two common organic pollutants in pharmaceutical industry wastewater streams (Kilroy and Gray 1992).The experiments employed continuous flow culturetechniques.

Materials and methods

Culture and medium

A mixed enrichment culture derived from a pharmaceutical indus-try wastewater treatment plant was used. Enrichment involvedmethanol and acetone as binary carbon energy substrates. The me-dium used was that of Carden and Hamer (1997) for continuouscultures.

Bioreactor

The bioreactor was a 14 dm3 (total volume) cylindrical stainlesssteel vessel fitted with four wall baffles, dual turbine impellers ona single shaft for agitation, an air sparger, and provided with bothtemperature and pH control and both medium inlet and cultureoutlet pumps, the latter attached to an overflow weir. In addition,the bioreactor was provided with a condenser on its air outlet. pHcontrol involved the addition of an equimolar 0.5 M NaOH/KOHsolution. The liquid volume, impeller speed, pH, temperature andsparged air-flow were maintained constant at 6.7 dm3, 900 rpm,6.7, 30°C and 4 dm3/min, respectively.

Analytical procedures

Optical density and dry weight were determined as described byCarden and Hamer (1997).

The concentrations of methanol and acetone in aqueous sam-ples were determined by gas chromatography using a modelHP489OA gas chromatograph (Hewlett-Packard, Dublin) fittedwith a 15 m HP-5 (crosslinked 5% PHME silicone) capillary col-umn with a film thickness of 1.5 µm and a flame ionisation detec-tor. Helium was used as the carrier gas and methyl isobutyl ketonewas used as an internal standard. Integration of the chromatogram-mes was performed using a model HP3395 integrator (Hewlett-Packard).

Lithium chloride concentrations were calculated using theflowing equations for well mixed continuous flow reactors.Lithium chloride added in feed:

C=Cin-Cine-Dt

Lithium chloride added as a pulse:

C=C0e-Dt

where C = concentration, D = dilution rate, t = time, subscript in =reactor influent, subscript 0 = zero time.

Results

Two types of experiments were carried out in continuousflow bioreactors which, prior to the imposition of chang-es in operating conditions, were operating as chemostatsat preselected dilution rates (i.e. 0.05, 0.15 and 0.2 h–1).The dual substrates, methanol (1 g/dm3) and acetone(1 g/dm3) were both completely utilised, giving a steadystate dry bacterial biomass concentration of about0.8 g/dm3. Experiments involved either the direct injec-tion of a LiCl pulse into the bioreactor or the introduc-tion of LiCl as an additional component in the continu-ous medium feed to the bioreactor. Both pulse additionsof LiCl and continuous feed concentrations of LiCl wereinvestigated and, in the latter, when the concentration ofLiCl in the culture approached that in the feed, the feedwas switched to a LiCl-free medium.

The first continuous flow experiments were conduct-ed at a dilution rate of 0.15 h–1 and involved determina-tion of the effects of a 10 g/dm3 LiCl pulse (Fig. 1a) anda switch to a feed containing 10 g/dm3 LiCl, followed bya switch to a LiCl-free feed (Fig. 2a). The pulse was in-troduced 1 h after initiation of the experiment, when thesteady state dry biomass concentration was 0.78 g/dm3

and both methanol and acetone were below detectablelimits. Biomass was washed out for approximately 25 h until a minimum dry biomass concentration of0.60 g/dm3 was reached. For the next 9 h, the dry bio-mass concentration remained erratic, but ultimately in-creased to 0.90 g/dm3 some 19 h after the pulse. There-after, a further reduction in the dry biomass occurred until a new steady state concentration of 0.80 g/dm3 wasattained around 24 h after the pulse. This was severalhours prior to complete LiCl washout. In addition, some4 h after the pulse, acetone built up in the culture andreached a maximum 6 h later. The acetone concentrationthen declined until about 15 h after build-up occurred,when its concentration fell below the detection level. In the case of LiCl incorporation in, and subsequent re-moval from the bioreactor feed, build-up and washouteach took approximately 30 h. No accumulation of eithermethanol or acetone occurred during 60 h, but for thefirst 10 h of LiCl build-up, the dry biomass concentra-tion decreased from 0.88 g/dm3 to about 0.70 g/dm3. After cessation of the LiCl feed, the dry biomass concen-tration gradually built up to 0.83 g/dm3. Clearly, the LiClpulse was more disruptive to the methanol/acetone-de-grading mixed culture than was the gradual change inLiCl resulting from its incorporation into the feed. Inboth cases, the presence of LiCl clearly reduced the carbon substrate-based biomass yield coefficient.

In order to determine the generality of the observedeffects, the impact of changes on both lower and higherculture dilution rates were examined. In the case of a10 g/dm3 LiCl pulse at a dilution rate of 0.05 h–1, an in-stantaneous drop in the dry biomass concentration from0.65 to 0.49 g/dm3 occurred, but no accumulation of either methanol or acetone was observed. As the LiClpulse was washed out, the dry biomass concentration in-

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creased to a new steady state of 0.69 g/dm3 after about30 h. Washout of the LiCl pulse took around 80 h. In thecase of a 10 g/dm3 on/off feed sequence of LiCl, LiClbuilt up to a concentration of 9.5 g/dm3 over about 72 hand then to near washout over 78 h. Neither methanolnor acetone accumulation occurred at any time duringthe experiment. However, after an initial drop in the drybiomass concentration from 0.67 g/dm3 to around0.61 g/dm3, the dry biomass concentration showed agradual increase to ca. 0.83 g/dm3 during LiCl accumu-lation and to around 0.94 g/dm3 during the first 32 h of LiCl washout. Clearly, at the lower dilution rate theculture was better able to accommodate the imposedchanges with respect to LiCl additions.

In order to examine the possible effects at a higher dilution rate (lower residence time), a further series ofexperiments concerning LiCl additions was conducted ata dilution rate of 0.2 h–1. In the case of a pulse additionof 10 g/dm3 LiCl (Fig. 1b), a drastic reduction in the drybiomass concentration from 0.88 to 0.45 g/dm3 occurredwithin 3.5 h. Subsequently, as the LiCl washed out overthe next 11 h, the dry biomass concentration increased to0.93 g/dm3. At the time at which the biomass reached itsminimum concentration, acetone started to accumulate,reaching a concentration of 0.15 g/dm3 after 8 h had

elapsed, only to decrease again after 11 h had elapsed, finally returning to below detection level concentrationsafter 12.5 h. The addition to and removal of LiCl fromthe feed (Fig. 2b) resulted in the most dramatic effects.Throughout LiCl build-up over 25 h, a reduction in thedry biomass concentration from 0.90 to 0.56 g/dm3 oc-curred. After 8 h had elapsed, first acetone and, subse-quently, methanol built up, the latter much more abruptlythan the former. Upon switching to the LiCl-free feed, afurther decrease in dry biomass to 0.48 g/dm3 occurred,after which it increased, peaking around 21 h later at1.06 g/dm3 and subsequently reaching a steady state con-centration of 0.95 g/dm3. After the switch, both methanoland acetone continued to accumulate further, reachingconcentrations of 0.41 and 0.36 g/dm3, respectively, after1.5 h and 10 h had elapsed from the switch, respectively.Elimination of the accumulated methanol and acetonethen occurred at rates exceeding those possible from thesum possible by stripping and washout effects, thus indi-cating degradation by the process culture. In this case,the effects of LiCl on both the biomass concentrationand the biomass yield coefficient were marked, but per-haps more important were the process instability thatwas introduced and the reduced performance concerningboth methanol and acetone elimination that occurred.

Fig. 1 Effects of 10 g/dm3

LiCl pulses on a chemostat operating at a substrate feedconcentration of 1 g/dm3 eachof methanol and acetone and atdilution rates of a 0.15 h–1 andb 0.2 h–1

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Fig. 2 Effects of 10 g/dm3

LiCl concentration in the feedof a chemostat operating at asubstrate feed concentration of1 g/dm3 each of methanol andacetone and at dilution rates ofa 0.15 h–1 and b 0.2 h–1

quirement for biotreatment processes. In addition, bio-mass recycle would damp process instability.

On the basis of the present study, the concentrationsat which Li+ can be expected to occur in industrialwastewater streams are unlikely to have significant adverse effects on the operation of aerobic wastewatertreatment processes. Even so, because of a lack of infor-mation concerning Li+ accumulation by aerobic bacteria,it would seem appropriate if Li+ elimination occurred atsource, rather than allowing Li+ release into compositeaqueous effluents. The present study did not identifywhether Li+ might become associated with the biomassor might remain in the clarified treated aqueous effluentthat would ultimately be discharged to nature.

Acknowledgements Thanks are due to Roche (Ireland), who pro-vided financial support for Maura O’Brien through the Irish–American Partnership Programme, to Pat O’Halloran, Dan Cash,Liam Morris, Tom Burke and Patricia Connelly for technical sup-port, and to Aoife Carney for processing this manuscript.

References

Carden AP, Hamer G (1997) Aerobic biotreatment of acetone andmethanol in a continuous flow bioreactor during steady stateoperation. Bioprocess Eng 16:119–125

Discussion

It seems probable that the effects of LiCl on bacterialgrowth stem from interference with transport rather than being of an osmotic nature. In the specific case of E. coli, Inaba et al. (1994) demonstrated thatNa+(Li+)/H+ antiporter systems were responsible for the detoxification of Li+. In the absence of evidence forother bacteria, one could assume that similar detoxifica-tion mechanisms occur. Antiport is one of three types ofsecondary active transport involving the simultaneoustransport of two materials in opposite directions acrossthe cytoplasmic membrane at the expense of an ion gra-dient established previously as a result of primary activetransport and involving the expenditure of energy. Suchenergy consumption might well be reflected duringgrowth in substrate-based biomass yield coefficient reduction, as observed in the present study.

LiCl did not prevent either methanol or acetone utili-sation by the process culture, but, where shorter resi-dence times were used, a significant disruption to pro-cess culture performance occurred. In some respects,substrate-based biomass yield coefficient reductioncould be an advantage because complete degradationwith reduced biomass production is an important re-

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