Characterization of an exopolysaccharide mutant of Nostoc spongiaeforme:Zn2++-sorption and uptake

Neetu Singh, R.K. Asthana and S.P. Singh*Algal Research Laboratory, Centre of Advanced Study in Botany, Department of Botany, Banaras Hindu University,Varanasi 221 005, India*Author for correspondence: Tel.: þ91-542-2307146/47, Fax: þ91-542-2368174, E-mail: sps@banaras.ernet.in

Received 30 October 2002; accepted 6 June 2003

Keywords: Exopolysaccharide, Nostoc spongiaeforme, variant, zinc

Summary

Exposure of the exopolysaccharide (EPS)-synthesizing cyanobacterium Nostoc spongiaeforme to Zn2+ (20 lM)transformed the biomass into white debris. However, a few blue–green pin-heads emerged after 2 weeks in the sameZn2+-containing medium and formed less mucoid microcolonies (1–2 mm) relative to the protruding colonies (2–4 mm) of the parent strain on nutrient agar. One of such survivors (designated as Zn20) that was stable through 10successive transfers in Zn2+-lacking medium has been adopted for further characterization. The parent strainretained almost 88% of the total EPS synthesized, the rest being released into the ambient medium, while for Zn20,the EPS retained approximated to 74%. Although the Zn2+-sensitivity of the mutant was comparable with that ofthe parent (LD50, 7 lM), Zn2+ uptake was still 5-fold higher in the former (2 lg mg)1 biomass dry wt., 20 lM,external concentration). Also, both the strains showed insignificant difference in Zn2+-sorption onto their isolatedEPS. The mutant was characterized by having higher cell carbohydrate content (642.8 lg mg)1 dry wt.) than itsparent (513.6 lg). The X-ray diffraction pattern revealed Zn2+ deposition on EPS from the parent mainly as zinchypophosphite monohydrate [Zn(H2PO2)2ÆH2O], whereas there was a lack of distinct peaks in similar samples fromZn20, thus confirming the amorphous nature. There was participation in Zn2+ binding of only COO), N@O, NO2,SO2 groups in the parent while participation of PAO and C@O groups in mutant EPS was evident in IR spectra.The observations suggest that the mutant could be deployed to achieve sustained EPS synthesis, its release andmetal sorption/desorption in repeated cycles.

Introduction

Cyanobacteria synthesize and also release their polysac-charide in a species-specific manner. A recent upsurge ininterest has led to the screening of cyanobacteria forlarge scale production and release of water-solublepolysaccharides (RPS) for various industrial applica-tions (De Philippis et al. 1998; 2000; Nicolaus et al.1999), including metal complexation/removal (Guptaet al. 2000; Shah et al. 2000) owing mainly to theabundance of uronic acid and carbonyl groups (Urrutia1997; De Philippis & Vincenzini 1998). Our earlierobservations indicated that cyanobacterial/bacterialgenera with copious exopolysaccharide (EPS), wereefficient metal sorbents (Chatterjee et al. 1996). Theamphipathic biopolymers from Acinetobacter lwoffiiRAG1 produced oil-in-water ‘emulsan’ for effectivemetal sorption/recovery (Gutnick & Bach 2000) andeffective metal sorption by EPS from Pseudomonasaeruginosa (Kazy et al. 2002). However, X-ray studieson sheaths from the cyanobacterium Phormidium uncin-

atum revealed a fibrillar component mainly composed ofhomoglucan with properties very similar but not iden-tical to those of cellulose (Hoiczyk 1998). As EPSsynthesis is also regulated by various environmentalregimes, efforts are needed to select cyanobacterialstrains that are constitutive for EPS synthesis and alsofor its release during growth as methods available toextract EPS in the ‘cell-bound’ category, are time-consuming and uneconomical (Reddy et al. 1996; Singhet al. 1999). The dominance of cyanobacteria in waterswith elevated Zn2+ levels and the emergence of tolerantstrains is well documented (Shehata & Whitton 1982;Whitton 1984). The sequestration of Zn2+ in polyphos-phate reserves (Jensen et al. 1982) or metallothionein(Daniels et al. 1998) is also on record. There areobservations that Zn2+-rich environments selected fornon-mucoid strains of Rhizobium leguminosarum (Pur-chase et al. 1997) and the mechanism of Zn2+ resistancein Pseudomonas putida has been observed (Choudhury &Srivastava 2001). However, the question remains wheth-er non-mucoid strains were defective in EPS production

World Journal of Microbiology & Biotechnology 19: 851–857, 2003. 851� 2003 Kluwer Academic Publishers. Printed in the Netherlands.

or whether a major fraction of EPS got liberated into theambient medium, thus leaving marginal amounts of‘cell-bound’ EPS.The present report examines a Zn2+-induced EPS

mutant of the cyanobacterium Nostoc spongiaeforme interms of EPS synthesis, its release, Zn2+-sensitivity/uptake and cation binding onto the isolated EPS basedon X-ray diffraction and IR spectra.

Materials and methods

Organism and culture conditions

The organism used in the present study was Nostocspongiaeforme Agardh ex Born. et Flah., a local isolatefrom a rice field, and its EPS mutant (Zn20). Cells weregrown diazotrophically in modified Chu-10 medium(Gerloff et al. 1950) lacking any combined nitrogensource, under cool white fluorescent illumination(14.4 W m)2) with a 18:6 h light/dark cycle at24 ± 1 �C. The nutrient solution contained macroele-ments (g l)1) : MgSO4 Æ 7H2O, 0.025; Na2CO3, 0.020;Na2SiO3 Æ 5H2O, 0.044; CaCl2 Æ 2H2O, 0.0735; K2HPO4,0.01; Fe-citrate, 0.0035; citric acid, 0.0035 and micro-elements (mg l)1) : H3BO3, 0.50; ZnSO4 Æ 7H2O, 0.05;MnCl2 Æ 4H2O, 0.05; CuSO4 Æ 5H2O, 0.02; MoO3, 0.01;and CoCl2, 0.04. All the chemicals were laboratorygrade products of Glaxo India Ltd., Mumbai, India.

Isolation of exopolysaccharide mutant

In preliminary trials, 15 lM Zn2+ was lethal. WhenN. spongiaeforme cells (initial density 75 lg proteinml)1) were dosed with 15 and 20 lM Zn2+, there wasefflux of phycocyanin pigment following 1 h leavingwhite biomass debris. Prolonged incubation up to (2–3 weeks) of the biomass in the same Zn2+-medium(20 lM) showed emergence of a few pin heads (4–10).Such pin heads grew in size when shifted to fresh Zn2+-lacking medium. One such strain (Zn20) was stablethrough ten successive transfers in Zn2+-lacking medi-um and was adopted for comparison with its parent.

Protein estimation

Protein was estimated by the Lowry method as modifiedby Herbert et al. (1971) using bovine serum albumin(Sigma) as standard.

Zn2+ uptake assay

Exponential phase (6 day) cells of N. spongiaeforme(parent) and the mutant (Zn20) were concentrated,washed three times with sterile, double distilled waterand suspended in growth medium (cell density,200 lg protein ml)1) containing 20 lM Zn2+ for 1 h

and uptake was monitored in the light (14.4 W m)2) at24 ± 1 �C. Cells taken out at selected intervals wereconcentrated (3000 · g) and the pellet resuspended inEDTA (10 lM) to remove the EDTA-extractable Zn2+.Such washed cells were dried and digested in 1 ml ofHNO3:HClO4 mixture (10:1, v/v) in a boiling water bath(30 min) to ensure digestion and release of the associ-ated metal ions. Samples were diluted to 5 ml with tripleglass-distilled water. A further centrifugation removedany undigested material, and the resulting supernatantwas analysed for Zn2+ (lg mg)1 dry wt.) in PerkinElmer-model 2380 atomic absorption spectrophoto-meter at 213.8 nm.

EPS extraction and estimation

EPS was extracted by the procedure of Reddy et al.(1996). Cells were concentrated at room temperature(10,000 · g, 5 min) to collect the biomass pellet for theseparation of associated EPS. The resulting supernatantwas concentrated (10-fold) by evaporation (40 �C) forpropanol precipitation. The pelleted biomass suspensionin an appropriate volume of triple distilled water, wasstirred gently followed by centrifugation at 4 �C(10,000 · g, 10 min). The EPS released was mixed 1:1with chilled isopropanol for precipitation and theprecipitate washed (3–4 times) with isopropanol (50%,v/v) to remove the adhering salts in the medium. TheEPS extract was oven dried (37 �C) and acid hydrolysed(HCl, 2 M) at 100 �C (2 h) as suggested by Panoff et al.(1988). Appropriately diluted hydrolysate (in distilledwater) was analysed for glucose (lg) by the method ofDubois et al. (1956). Total EPS corresponded to theamount liberated after cold centrifugation of cyanobac-terial cells plus that already liberated into the ambientmedium. EPS concentration is expressed as glucoseequivalent mg)1 biomass dry weight.

Carbohydrate estimation

Total carbohydrate content of cyanobacterial cells wasassayed by phenol–sulphuric acid method (Dubois et al.1956).

Zn2+ binding onto the EPS

Zn2+ (20 lM, saturating level for uptake) binding ontothe EPS from both the strains (g l)1) was monitored(30 min) in deionized water containing 20 lM Zn2+.Following equilibration, two volume of ice cold isopro-panol were added to metal/EPS solution to precipitatethe polymer and then centrifuged (10,000 · g, 20 min).Amount of Zn2+ adsorbed onto the EPS was deter-mined by measuring the residual metal concentration inthe filtrate using the atomic absorption spectrophoto-meter (as described before) at 213.8 nm and expressedas lg mg)1 EPS.

852 N. Singh et al.

X-ray diffraction

The X-ray diffraction pattern of dried powder samplesof metal-free control and Zn2+-sorbed EPS materials ofboth the strains was recorded in Seifert 3000 P PowderDiffraction equipment using monochromatic CuKa

radiation (k ¼ 1.540598 A) over the range of 10–100�(2h) with a step length of 0.05� (2h). The identification ofZn2+salts is based on a comparison with the PowderDiffraction File Inorganic Volume No. PD1S-5iRB,JCPDS, 1845 Walnut Street Philadelphia, Pennsylvania,1967 and Organic Volume No. PD1S-50RB JCPDS,1960.

FTIR spectroscopy

Infrared spectra of dried polysaccharide samples (bothcontrol and Zn2+-loaded) of both the strains wererecorded in a JASCO 5300 FTIR spectrometer equippedwith laser detector. The samples were prepared as KBrdiscs.

Statistical analysis

All experiments were carried out in triplicate withstandard errors represented as bars wherever necessary.

Results

Colonial morphology

Nostoc spongiaeforme formed convex colonies (2–4 mmdia., average) with copious polysaccharide. The mutant(Zn20) in contrast, was sluggish in growth (Table 1) thatresulted in production of less mucoid microcolonies (1–2 mm dia., average) during a comparable time(20 days).

EPS production

The apparent variation in colony shape/size tempted usto investigate whether there was also alteration in thesynthesis of EPS by the mutant or in the level of itsZn2+-sensitivity/uptake relative to the parent. A timecourse (0–16 days) of the extent of ‘bound’ and ‘re-

leased’ EPS in variant and its parent counterpartpresented in Figure 1, revealed the initial jump (0–4 days) in the amount of EPS released by the mutant,although cells lacked the initial background. The risingtrend for this category of EPS continued until day 16 toraise it to 17.43 lg glucose, thus presenting a contrastwith the parent as evident from only a marginal increase(2.9 lg glucose, after 16 days). Therefore, such a highrate of EPS release by the mutant cells was perhaps thereason for a slow increase in viscosity of liquid cultureswith passage of growth.

Zn2+ binding in intact cells

The Zn2+-sensitivity of Zn20 was comparable with itsparent. Zn2+ uptake however, increased almost 5-fold(2 lg Zn2+ mg)1 dry wt., after 1 h) over the parent atthe common external concentration of 20 lM (Fig-ure 2). Zn2+ uptake for the parent saturated beyond5 min, while for Zn20, it was beyond 30 min.Table 1 provides a comparison of all the above

parameters in respect of the parent and Zn20 withadditional observations on liquid growth and cellcarbohydrate content of the respective strains. Zn20was slow growing, as evident by 25% reduction in

Table 1. Comparison of cell carbohydrate, selected parameters in parent and mutant (Zn20) of Nostoc spongiaeforme (diazotrophic growth).

Parent Mutant (Zn20)

Colony size (nutrient agar) Large (2–4 mm) circular (Spherical) Small (1–2 mm) circular (Spherical)

Filaments Slightly spiral, mucoid Straight, less mucoid

Growth yield (–N, 16 days) protein (lg ml)1) 196 ± 6 157.5 ± 5.5

EPS (bound) (16 days) 88% 26%

EPS released (16 days) 10–12% 74%

Cell carbohydrate (lg mg)1 dry wt, 16 days) 513.6 ± 15.4 642.8 ± 19.5

Zn2+ sensitivity Sensitive (LD50, 7 lM) Sensitive (LD50, 7 lM)

Zn2+ uptake ext. conc. (20 lM) (lg mg)1 dry wt, 30 min) Low 0.41 ± 0.016 High 2.0 ± 0.08

Zn2+ sorption (lg mg)1 EPS) 0.877 ± 0.026 0.673 ± 0.024

Figure 1. Relative amounts of bound and liberated EPS in the parent

and Zn20 strain of N. spongiaeforme. Bound EPS-(parent) (s) and

Zn20 (j); released EPS-(parent) (h) and Zn20 (d).

Nostoc Zn2+-uptake mutant 853

growth yield (157.5 lg protein ml)1) but had a highercarbohydrate content (642.8 lg glucose) than its parent(513.6 lg glucose). Such a variation has been implicatedin the differential degree of the diversion of cellcarbohydrate towards EPS synthesis in addition toother metabolic requirements. Zn2+-binding onto theEPS isolated from the parent (0.877 lg mg)1) and Zn20(0.673 lg mg)1) showed insignificant difference.

Zn2+-binding onto the isolated EPS (X-RD and IRspectra)

An X-ray diffraction study of EPS in metal-less controland those exposed to Zn2+ was carried out to ascertainthe chemical nature of the sorbed metal. The diffracto-gram for Zn2+-less EPS as control from the parentcharacterized a fibrillar component with a substantialdegree of crystallinity and maximum number of peakscorresponding to 2h of 33.7�, 36.4� and 43.8� whereinthe D-value in most cases corresponded to dextrose(Figure 3a). By comparison, the EPS of Zn20 (Zn

2+-less)showed maximum peaks at 2h of 31.6�, 32.2�, 33.8�,43.8� and 49.8�; the D-value indicating dextrose. Otherpeaks at 2h (15.2� and 23.8�) had D-value correspondingto fructose with the exception of peaks at 2h of 21.2� formannose (Figure 3c). However, the spectrum for Zn2+-loaded EPS of the parent showed several sharp peaksreflecting the deposition of Zn2+ crystals (peaks at 2hof 29.6�, 36.2�, 39.6�, 43.4�, 47.7� and 48.8�) withD-value reflecting zinc hypophosphite monohydrate[Zn(H2PO2)2 Æ H2O] (Figure 3b). By contrast, Zn2+-loaded EPS from Zn20 lacked any distinct peak in away characterizing its amorphous nature or in otherwords, binding of metal was not sequential i.e., non-systematic arrangement (Figure 3d).The FTIR spectra (400–4000 cm)1) of native and

metal-loaded EPS of the respective strains are shown inFigure 4a–d. For EPS native to parent strain, there weredistinct sharp stretching frequencies at �2920, 2890 and

1583, 1548 cm)1 indicating the presence of CAH stretchand COO) asymmetrical stretch, respectively. Otherstretching vibrations at 1498, 1454, 1388, 1280 and1150 cm)1 indicate the presence of N@O stretch, CAHbend, NO2 symmetrical stretch and SO2 symmetricalstretch, respectively (Figure 4a). However, the Zn2+-loaded EPS revealed disappearance of the peaks at�1583, 1548, 1498, 1454, 1280 and 1150 cm)1 indicatingCOO), COO) asymmetrical stretching, N@O, N@Ostretching, NO2 symmetrical stretching, and SO2 sym-metrical stretching as a consequence of Zn2+ sorption(Figure 4b).For Zn20 EPS, there were distinct sharp stretching

frequencies at �2920, 1740, 1660, 1601, 1514, 1467,1310, 1250, 1145 and 1018 cm)1 indicating CAH, C@O,C@C stretch, COO) asymmetrical stretch, NO2 asym-metrical stretch, N@O stretch, SO2 asymmetricalstretch, NO2 symmetrical stretch, SO2 symmetricalstretch and PAO stretch, respectively (Figure 4c). Acomparison of spectra for Zn2+-loaded EPS of Zn20indicates characteristic blue shift (negative shift) in theSO2 symmetrical stretch, possibly as a result of interac-tion of Zn2+ with SO2 groups while a positive shift inPAO stretch could be due to PAO group. The disap-

Figure 2. Zn2+ uptake (20 lM, external concentration) by parent

N. spongiaeforme (s) and Zn20 strain (d).

Figure 3. X-ray diffraction spectra for (a) parent N. spongiaeforme

EPS as control (lacking-Zn2+) (b) Zn2+-loaded, (c) mutant (Zn20) EPS

as control (lacking-Zn2+) and (d) Zn2+-loaded.

854 N. Singh et al.

pearance of peaks at 1740, 1601, 1514, 1310, 1250 cm)1

for Zn2+ indicates C@O, asymmetrical stretching forCOO), NO2, SO2 and NO2 symmetrical stretching as aconsequence of Zn2+ sorption (Figure 4d).

Discussion

Cyanobacteria dominate the Zn2+-enriched waters ofelevated pH (Whitton 1980) and Zn2+ is a micronutrientfor general cyanobacterial growth and production ofspecific metabolites (Lukac & Agerter 1993). Thecontribution of mine drainage and industrial processes

in contamination of many lakes and estuaries by Zn2+ iswell documented (Say & Whitton 1981; Forstner 1983).Cyanobacteria sequester cations like Cu2+, Zn2+, andNi2+ in their polyphosphate reserve (Jensen et al. 1982),and metallothionein (MT) complexes with Zn2+ ions(Daniels et al. 1998). There are reports that wild typeunicellular Anacystis nidulans tolerated Zn2+ up to0.1 mg l)1, while its tolerant strain could go up to1 mg l)1 accompanied by a reduced Zn2+ uptake,indicating that uptake was not linked with tolerance(Shehata & Whitton 1982).Whether the small amount of EPS in Zn20 was also

associated with increased tolerance to Zn2+, was not thecase, as evident from the sensitivity test (Figure 1).Similarly, the lowered amount of EPS (cell bound) inZn20 did not accompany lowered Zn2+ uptake, and thedata formed a contrast as Zn2+ uptake increased almost5-fold (2 lg Zn2+ mg)1 dry wt., 1 h) over the parent(Figure 2).In general, EPS production in cyanobacteria and

bacteria is growth-dependent, and the polymer beinganionic, often bioconcentrates metal cations, restrictstheir entry to the cell interior, and in the latter case,increased polymer production has been associated withmetal tolerance (McLean et al. 1996). The presentobservations on high Zn2+ uptake by Zn20 are inline with the recent report of a 3-fold Cd2+ uptake bynon-mucoid strains of Rhizobium leguminosarum thatwas associated with high levels of cell carbohydrate(Purchase et al. 1997). A comparison in Table 1 alsoascertained that mutant cells had a carbohydrate con-tent 1.3-fold (642.8 lg glucose equivalent) relative to theparent (513.6 lg). Interestingly, however, a mere ap-prox. 1.3-fold enhancement in the cell carbohydrate inZn20 could eventually lead to an approx. 5-fold rise inZn2+ accumulation. The obvious reason put forward forcell carbohydrate-associated Zn2+ uptake could be itshydrophilic nature, leading to increased cation immobi-lization at the cell interior (Purchase et al. 1997) . Incyanobacteria, changes in cell carbohydrate are linkedwith the N-budget in the marine environment (Mitsui &Cao 1988), and in slow growing cyanobacteria like thepresently used mutant, the high rate of carbohydratesynthesis was possibly to meet the C-demand duringdiazotrophic growth, along with a major part beingdiverted towards synthesis of EPS (Stal 1995).Studies based on the actual cell surface sites chemi-

cally involved with metal binding are rare (Banfield &Nealson 1997). However, Philip et al. (1995) obtainedhigher Cu biosorption capacity, i.e., 50 mg g)1 dry cellsfor P. aeruginosa. Kapoor et al. (1999) applied theLangmuir and Freundlich models for metal sorption byAspergillus niger. In a recent report, Micrococcus luteusIAM 1056 had an appreciable capacity to bind/adsorbCu (Nakajima et al. 2001). The present investigationinvolved EPS isolated from both parent and a mutantstrain of N. spongiaeforme to ascertain the chemicalnature of Zn2+-EPS interaction (Figure 3a–d). Thesharp, distinct peaks for the Zn2+-loaded samples

Figure 4. FTIR spectra for (a) parent N. spongiaeforme EPS as control

(lacking-Zn2+), (b) Zn2+-loaded, (c) mutant (Zn20) EPS as control

(lacking-Zn2+) and (d) Zn2+-loaded.

Nostoc Zn2+-uptake mutant 855

confirmed Zn2+-sorption onto the EPS. According toHoiczyk (1998), X-ray diffraction of the sheath of thefilamentous cyanobacterium Phormidium uncinatumsuggested that the fibrillar component was a homoglu-can, very similar but not identical to cellulose in beingcross-linked with other monosaccharides. Our observa-tions indicate the presence of dextrose, based on peakswith characteristic D-value in the case of EPS from theparent (Figure 3a) while for its mutant, the addition offructose and mannose as deciphered from peaks withcharacteristic D-values (Figure 3c). Whereas Zn2+-load-ing of EPS from the parent formed zinc phosphite salts(Figure 3b), the same target from Zn20 lacked anydistinct peaks in a way suggesting asystematic arrange-ment of the cation sorbed (Figure 3d).A comparison in Figure 4a–d accounts for the fate of

relative groups on the EPS from parent and mutant(Zn20) following Zn2+ complexation. Binding of metalson the bacterial surface results from interaction withvarious anionic ligands like S2), HCO�

3 , CO2�3 , SO2�

4 ,OH) or PO3�

4 in the lipopolysaccharide and othersurface polymers of Gram negative bacteria (Beveridge1981; Fortin et al. 1995) including other detoxifyingligands (Kurek et al. 1991; Nies 1992; Volesky &Prasetyo 1994). In the B. subtilis cell wall, metal bind-ing occurs primarily by complexation with availableconstituent carboxylate and phosphodiester groups(Beveridge et al. 1983). The reactive chemical groups,carboxylate and phosphate exposed at the cell surface,complex with metals to form metal precipitates thatgrow at the expense of available counter ions (OH),HCO�

3 , SO2�4 , etc.). Such precipitates are poorly ordered

and hydrous initially, but over time, lose water and turncrystalline (Fortin et al. 1995). The formation of metalphosphites is also reported as a result of bacteria-metalinteraction (Macaskie et al. 1992; Nakajima et al. 2001).The oxygen and nitrogen donor atoms from the carb-oxyl and amino groups play a vital role in Cu-sorptionby the A. niger and B. subtilis cell walls (Beveridge &Murray, 1980; Kapoor et al. 1999).The overall observations show the stability of Zn20 in

liberating EPS into the ambient medium indicating thatsuch a Zn2+-induced genetic change was favourable toits commercial exploitation in high EPS yield throughrepeated growth cycles. And in turn, the isolatedpolymer can be applied for large scale metal removalin a cost-effective way.

Acknowledgements

This work was supported by a grant from the Ministryof Environment and Forests, Government of India(grant no. 19/30/95-RE dt. 24 August, 1998) andCouncil of Scientific and Industrial Research, NewDelhi. The availability of the X-ray diffraction facility atIIT, Roorkee and the FTIR facility at the Departmentof Chemistry, Banaras Hindu University, India isgratefully acknowledged.

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