Separation and Purification Technology 62 (2008) 337–341

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Separation and Purification Technology

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Nickel inhibition of calcium precipitation by ureolyticmixed microorganisms under batch conditions

Mustafa Isik ∗

0 Aksa

ent o) by utch ce BOed a zof Nireduk0), auringthe kiemovuringconce

Aksaray University, Engineering Faculty, Environmental Engineering Department, 6810

a r t i c l e i n f o

Article history:Received 2 January 2008Received in revised form 2 February 2008Accepted 8 February 2008

Keywords:UreolyticCalciumInhibitionNi(II)Mixed

a b s t r a c t

The respirometric assessmbonate precipitation (MCPmineral medium under ba224 mg Ni(II) L−1 from thsubstrate removal rate fittthe last period, for a rangetions from 0 to 512 mg L−1

zero-order rate constant (first-order reaction rates dtion in order to determineremoval rate (Rmax). BOD ring Ni(II) concentrations dobserved at a higher Ni(II)

1. Introduction

Water containing high concentration of calcium tends todeposits scale on the inside of pipes and on the appliances duringwater use. Also, calcium-rich water and wastewater is one of theproblems in the water and wastewater treatment processes. Pre-cipitates of calcium are associated with young landfill leachates,reverse osmosis concentrates, industrial processes such as boneprocessing, paper recycling, and sugar processing [1–3] and indus-trial wastewaters containing lime, which is used as an inexpensiveneutralization [4]. Existing classic chemical crystallization reac-tors are based on the addition of a base [NaOH or Ca(OH)2] inthe presence of nucleation site (e.g. sand grains). Such reactorsare, however, often expensive, complex, and sometimes give riseto highly alkaline effluent [5] requiring neutralization before bio-logical treatment.

A new biological approach for calcium removal from calcium-rich industrial wastewater was also reported by a research groupwho proposed microbial carbonate precipitation (MCP) processbased on microbial urea hydrolization [3,5–7]. Microorganismshave long been known to catalyze the precipitation of CaCO3 in

∗ Tel.: +90 382 2150953/132; fax: +90 382 2150592.E-mail address: mustafaisik@nigde.edu.tr.

1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.seppur.2008.02.002

ray, Turkey

f the inhibitory impact of Ni(II) on substrate utilization and microbial car-reolytic mixed microorganisms was investigated with a glucose containingonditions over an incubation period of 134 h. The IC25 was determined asD values of samples. The interpretation of kinetic data showed that theero-order at the beginning of the incubation period and first-order during(II) concentrations between 0 and 512 mg L−1. Increasing Ni(II) concentra-ced the substrate degradation rate constant from 10.8 to 5.3 mg L−1 h−1 fornd from 0.015 to 0.002 h−1 for first-order rate constant (k1). The zero- andincubation period were equalized to the reaction rate of the Monod equa-

netic constants, half saturation concentration (KS) and maximum substrateal rate was inhibited accordingly to mixed inhibition model with increas-the calcification process. Also, the inhibition of calcium precipitation wasntration because of inhibition of ammonium production in these samples.

© 2008 Elsevier B.V. All rights reserved.

natural environments such as oceans, soils and saline lakes, in aprocess referred to as MCP [8,9]. The generally accepted mecha-nism of MCP is the increase in pH and dissolved inorganic carbon(DIC) of a given environment through normal physiological activi-ties [8]. Under aerobic conditions, one of the known MCP processes

is heterotrophic microbial urea hydrolysis processes, in which 1 molof urea is hydrolyzed by the urease enzyme to 2 mol of ammoniumand 1 mol of carbon dioxide. These products can subsequently reactto form ammonium and carbonate ions, which, in the presenceof soluble calcium ions, can react and precipitate as CaCO3. Ureahydrolysis provides simultaneously a pH and CO2 increase, both ofwhich are responsible of CaCO3 production. In addition to thesefactors, precipitation process of CaCO3 needs nucleation site, e.g.sand, suspended solids, and bacteria in the medium.

The input of heavy metals into the aquatic environment hasincreased parallel with the rapid industrialization of our world dur-ing the past 150 years. Heavy metals can be stimulatory, inhibitory,or even toxic in biochemical reactions depending on their concen-trations. A trace level of many metals is required for activation orfunction of many enzymes and co-enzymes. Excessive amounts,however, can cause inhibition or toxicity. This is mostly due to thechemical binding of heavy metals to the enzymes, resulting in thedisruption of enzyme structure and activities [10,11]. Many indus-tries, such as automotive, metal producing, electroplating, batterymanufacturing, mining, electric cable manufacturing, tannery, steeland textile, release various heavy metals like nickel, cadmium, lead,

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338 M. Isik / Separation and Purific

chromium and copper in wastewaters. These heavy metals are toxicto aquatic ecosystems and human health, get accumulated in organ-isms beyond tolerance levels [12] and are also non-competitiveinhibitors to substrate utilization [13]. A number of methods havebeen proposed for measuring metal toxicity in biological systems.The more commonly used ones include the measurement of enzy-matic activity, the measurement of respiratory rate, influence onthe microorganism growth parameters and the use of fluorescentand bioluminescence methods [14].

Ni(II) is a heavy metal frequently occurring in raw wastewa-ter streams from industries such as non-ferrous metal, mineralprocessing, paint formulation, electroplating, porcelain enamel-ing, copper sulphate manufacturing and steam-electric powerplants [15]. While the Ni(II) ion concentration in plating rinse canapproach 2–900 mg L−1, wastewater from paint and ink formula-tion, porcelain enameling, copper sulphate manufacture industriesrecord effluent Ni(II) ion concentrations that varies from 0–40,0.25–67 and around 22 mg L−1, respectively [16]. Ni(II) whichbelongs to the so-called “essential” metals has been identified asa component in a number of enzymes, participating in impor-tant metabolic reactions, such as: ureolysis, hydrogen metabolism,methane biogenesis and acidogenesis [14].

Calcium removal from industrial wastewater is a new proposedmethod and it has not been studied extensively by consideringenvironmental variables. In this study, the inhibitory effect of Ni(II)on ureolytic microbial community was studied by evaluating BODchange and Ca precipitation under batch conditions.

2. Materials and methods

2.1. Synthetic wastewater

The synthetic wastewater was prepared by considering thecomposition of liner paper manufacturing and medium strengthmunicipal wastewater proposed by Kim et al. [17], and Holakoo etal. [18], respectively. The wastewater contained (mg L−1): glucose-COD (750); urea (600); CaCl2 (17); MgSO4·7H2O (1541); KH2PO4(132); FeCl3·6H2O (19); CuSO4·5H2O (0.118); MnSO4·H2O (0.123);ZnCl2 (0.229), CoCl2·6H2O (0.404); Na2CO3 (477); NaHCO3 (378).Sulphuric acid was used to maintain a pH of 7.00 ± 0.10. Compo-sition of synthetic wastewater resulted in glucose-COD/N/P ratioof 100/37/4. Urea was used in a higher concentration than what isnecessary for growth in the medium to serve MCP process. CaCl2

2+ −1

was also added to the medium as total Ca of 400 mg L duringbatch experiments.

2.2. Sludge production and experimental setup

A mixed culture was obtained from a fed-batch reactor receivingthe synthetic wastewater described above and used as the initialinoculum for the batch experiments. The sludge retention timeand biomass concentration in this reactor were approximately 10 dand 2000 mg L−1 as mixed liquor volatile suspended solids (VSS),respectively. Dissolved oxygen was measured above 2 mg L−1. Thesludge can convert of 0.6 g urea L−1 d−1 to ammonia. The producedsludge had a VSS/suspended solids (SS) ratio of 0.82 and a sludgevolume index (SVI) of 92 mL g−1.

The experiments performed in duplicate, were carried out todetermine the Ni(II) inhibition to urea hydrolyzing bacteria via BODmeasurement during the period and change of other parameterssuch as, calcium, alkalinity, pH, SS, and VSS after experimen-tal period of 134 h. Eighteen BOD bottles were used, twice foreach of the nine tested Ni(II) concentrations. The effective volumeand initial sludge concentration were 97 mL and 220 mg VSS L−1,

echnology 62 (2008) 337–341

respectively, in the bottles. Experiments were carried out in anincubator in which bottles were mixed with magnetic stirrer at20 ◦C.

2.3. Respiration–inhibition test

Respiration–inhibition tests based on BOD measurements werecarried out in bottles of WTW Oxi Top system. The syntheticwastewater containing the mineral medium given above was addedto bottles. Ni(II) concentration varying between 0 and 512 mg L−1

in the bottles were adjusted by stock solution of 800 mg Ni(II) L−1

as NiCl2·6H2O. Control samples were performed in the bottles con-taining no Ni(II). Oxygen consumption was monitored at specifiedtimes and compared to the control samples. Inhibition was definedas a decrease in oxygen consumption compared to the control sam-ples. The inhibitory effect of Ni(II) (percentage inhibition) at eachconcentration was calculated as

I(%) = RB − R24

RB× 100 (1)

Where RB and R24 are respiration rates of blank control, and ofthe tested concentration of Ni(II) for initial 24 h, respectively (mgBOD L−1 h−1). IC50 and IC25 are Ni(II) concentrations (mg L−1) elicit-ing a 50% and 25% inhibitory effect after 24 h, respectively, IC valueswere derived after plotting percentage inhibition against concen-tration and by assuming linearity.

2.4. Analytical methods

Samples were withdrawn from the mixed liquor medium afterincubation time, and were centrifuged at 2375 × g for 10 min toremove SS from the medium. Clear supernatants were analyzedfor alkalinity, ammonium, and Ca2+ concentrations of samples atthe end of the incubation period. Standard kit and spectrometricmethod described in the guide of Merck-Spectroquant were usedfor ammonium. VSS, SS, SVI, alkalinity and Ca2+ were analyzed asspecified in standard methods [19]. The pH and DO were measuredby using with WTW pH330i/SET and WTW Oxi340i/SET, Germany,respectively.

3. Results and discussion

3.1. Inhibition level of substrate removal

The effect of Ni(II) on the substrate removal was monitored usingBOD data by adding various concentrations of Ni(II) ions into theassay. The response of bacteria to higher Ni(II) concentration wasdepicted in Fig. 1. Ni(II) up to concentrations of 128 mg L−1 had noapparent effect on substrate removal. IC50 and IC25 were calculatedas 525 and 224 mg L−1 from the plot of Ni(II) concentration ver-sus inhibition % (Fig. 1). Ni(II) showed an inhibitory effect on BODdegradation by ureolytic mixed microorganism in a concentration-dependent manner. For samples containing 0 and 512 mg Ni(II)L−1, the BOD values decreased from 250 to 140 mg L−, respec-tively, during the incubation period of 24 h. As substrate was almostconsumed by ureolytic mixed bacteria, the rate of substrate con-sumption gradually decreased with respect to Monod kinetic thatis clarifies substrate-limiting growth. BOD data clearly show thatNi(II) concentrations which are higher than 128 mg L−1 inhibitedthe substrate oxidation rate of ureolytic mixed bacteria.

The BOD values increased when the Ni(II) concentrationincreased from 0 to 64 mg L−1, and thereafter decreased graduallyto the lowest value (at concentrations higher than 128 mg L−1). Thedata indicates that Ni(II) stimulates the ureolytic mixed bacteriaat concentrations below approximately 64 mg L−1 while at higher

M. Isik / Separation and Purification T

of ammonia from urea, but did not exceed 8.59, as a result of theammonium buffer equilibrium and CaCO3 precipitation. Low pHvalues are one of the main advantages of bio-catalytic calcificationprocess compared to chemical ones.

The low ratio of the VSS/SS in the samples containing no orlower Ni(II) concentrations also indicated the precipitation of Ca2+

together with bacteria as shown in Fig. 3. The VSS remained moreor less constant in all samples, while SS was higher with no orlow (<16 mg L−1 Ni(II)) than with higher concentrations. The VSS/SSratio increased from 0.37 for Ni(II) free sample to 0.73 for the Ni(II)concentration of 512 mg L−1. Microscopic examination of the cal-cifying sludge showed the presence of small, but dense calcareousflocs, particularly taken from samples with no or lower Ni(II) (notshown). Also, calcareous flocs were observed with the naked eye.

3.3. Determination of kinetic coefficients

Fig. 1. BOD values during the incubation (a) and inhibition percentages after 24 h(b) of samples containing various Ni(II) concentrations (error bars show standarddeviations).

concentrations it acts as a growth inhibitor. A number of researchershave reported only the stimulatory effect of Ni(II) on aerobic mixedcultures. These studies showed the stimulatory effect of Ni(II) con-centrations such as, approximately up to 10 mg L−1 [20], 27 mg L−1

[14], and 40 mg L−1 [21]. Ainsworth et al. [22], who worked withbatch cultures of Klebsiella pneumoniae, concluded that an increaseof the Ni(II) concentration resulted, both, to prolonged lag times,and to a decrease of the final MLSS concentration. More specifically,they reported a reduction on the MLSS from 41.2 to 15.9 mg L−1

when the Ni(II) concentration in the growth medium was increased

from 300 to 500 mg L−1. They also reported a 50% reduction com-pared to a control sample in microbial survival for K. pneumoniae,by the use of viability counts on agar plates, at a Ni(II) concentrationof 6.65 mg L−1. For Ni(II), a research group [23] reported completegrowth inhibition between 20 and 30 mg L−1 for Nocardia corallina,at 30 mg L−1 for Bacillus brevis, between 40 and 50 mg L−1 for Bacil-lus megaterium, Pseudomonas aeruginosa, Nocardia rhodochrous andAsticcacaulis excentricus, at 50 mg L−1 for Serratia marcescens, andover 100 mg L−1 for Caulobacter leidyi. Kelly et al. [24], determinedthe EC50 values for activated sludge in the presence of Ni(II), bythe use of bioluminesence, to be over 100 mg L−1, while the samevalue was measured as 76 mg L−1, by the use of the specific oxygenuptake rate (SOUR) method. Although the above results are gener-ally comparable with the results obtained in the present study, itis suggested that the ureolytic mixed culture used in this study hashigher tolerance to Ni(II) than the ones cited in the literature.

3.2. Ca2+ removal

MCP process is based on microbial urea hydrolysis, thus, theincrease of the pH and ammonium values results in the precipita-

echnology 62 (2008) 337–341 339

Fig. 2. Alkalinity, soluble Ca2+, ammonium, and pH values after 134 h of incubationwith varying Ni(II) concentrations.

tion of soluble calcium in the medium. As shown in Fig. 2, whenNi(II) concentration was increased to 512 from 128 mg L−1, solu-ble calcium levels increased to 349 mg L−1, indicating that Ni(II)concentration >128 mg L−1 were inhibitory. Also, ammonium, alka-linity and pH levels decreased with Ni(II) at >128 mg L−1. Thesefindings clearly showed that ureolytic mixed culture was inhib-ited by Ni(II) ions. Urea was not effectively hydrolyzed by bacteria,and hence, pH, ammonium and alkalinity were low in the samplescontaining high levels of Ni(II). Calcium removal was strictly depen-dent on urea hydrolyzing products, such as, NH3, CO2 as reported byHammes et al. [5]. The pH increased with increasing the production

Traditionally, Monod type rate model has been widely used fordescribing the rate of degradation of a substrate by living cells:

−dS

dt= −R = RmaxS

KS + S(2)

Fig. 3. The effect of various Ni(II) concentrations on VSS, SS, and VSS/SS of sludgeafter 134 h of incubation.

ation Technology 62 (2008) 337–341

Fig. 4. (a) Zero-order reaction kinetics for BOD between 750 and 610 mg L−1 and (b)first-order reaction kinetics for BOD values between 610 and 490 mg L−1.

The KS and Rmax values were calculated based on the assumptionof S � KS (zero-order) and KS � S (first-order) in order to determine

340 M. Isik / Separation and Purific

where S (BOD) is substrate concentration (mg L−1), KS is half sat-uration concentration (mg L−1), R is substrate (BOD) utilizationrate (mg L−1 h−1), and Rmax is maximum substrate removal rate(mg L−1 h−1).

Inhibition models are classified according to the effect of toxiccompounds on Rmax and KS values. For example, depending on thetype of substances and its concentration the variations in Rmax andKS values and the inhibition are expressed by the following equationfor non-competitive model [13]:

−dS

dt= −R = Rmax

(1 + KS/S)(1 + IN/KI)

× (non-competitive inhibition model) (3)

where IN is inhibitor (Ni(II)) concentration (mg L−1), and KI is con-stant of inhibition (mg L−1).

Substrate removal rate is constant regardless of the substrateconcentration if S is significantly higher than KS. In this situation,the reaction order is zero as following [25]:

−dS

dt= k0 ∼= Rmax (4)

where k0 is zero-order rate constant through substrate removal(mg L−1 h−1).

The rate of degradation of substrate is first-order with respectto its concentration S, at low concentrations (KS � S) [25]:

k1S ∼= SRmax

KS(5)

KS ∼= Rmax

k1(6)

where k1 is first-order rate constant through substrate removal(h−1).

Since the Monod kinetic could not be applied (Monod kinetics

shows very low correlation, data not shown) to detect Rmax and KSthrough biodegradation, Equations given in Eqs. (4) and (6) wereused to determine the kinetic constants.

Rmax values were calculated from the substrate removal ratebetween BOD values 750 and 610 mg L−1 (Fig. 4) as substrateremoval rate was appropriate to zero-order kinetic model with thisperiod. After this period, substrate removal rate was assumed first-order containing Ni(II) concentrations between 0 and 512 mg L−1 upto BOD value of 490 mg L−1. Fig. 4 depicts zero- and first-order plotsin order to determine the kinetic constants for samples containingvarious Ni(II) concentrations. The kinetic and correlation coeffi-cients relevant to the zero- and first-order rate are summarizedin Table 1. Increases in Ni(II) concentration from 0 to 512 mg L−1

reduced the k0 values from 10.8 to 5.3 mg L−1h−1, and k1 valuesfrom 0.0153 to 0.0020 h−1 at the corresponding intervals.

For samples between without Ni(II) and concentration of512 mg L−1, reaction may be equalized to the zero-order sub-strate kinetic with respect to Eq. (4) during period in where BODdecreased to 600 from 750 mg L−1. After the period, reaction ratemay be equalized to the first-order to determine KS in Monodkinetic as shown in Eq. (6).

Table 1The kinetic constants obtained in aerobic batch tests during BOD degradation

Constant BOD interval (mg L−1) Ni(II) concentration (mg L−1)

0 4 8

k0 (mg L−1 h−1) 750–610 (zero-order) 10.8 9.7 9.4r2 0.988 0.989 0.976

k1 (h−1) 610–490 (first-order) 0.0153 0.0165 0.0215r2 0.986 0.979 0.992

the inhibition constants (KI). Rmax values were assumed approx-imately the same., hence, the average values were calculated fordata on which no inhibition were observed for Ni(II) concentra-tions up to 128 mg L−1. As a result of these calculations, the obtainedkinetic constants are given in Table 2 for samples with and withoutinhibition.

Fig. 5 clearly shows the increasing Ni(II) inhibition on sub-strate removal rate. The reciprocal fits showed that Ni(II) causedroughly non-competitive inhibition on BOD degradation resultingin increases in KS values and decreasing Rmax. The slope of the no

Table 2Maximum substrate utilization rate, half saturation concentration, and the valuesof inhibition constant

Constant Ni(II) concentration (mg L−1)

0–64 128 256 512

Rmax (mg L−1 h−1) 9.9 ± 0.7a 8.4 5.1 3.84KS (mg L−1) 502 ± 62 578 872 890KI for KS (mg L−1) Not applicable 842 230 120KI for Rmax (mg L−1) Not applicable 725 586 596

a Values ± show standard deviation.

16 32 64 128 256 512

10.5 10.1 8.8 8.4 6.9 5.30.981 0.982 0.981 0.985 0.976 0.977

0.0208 0.0252 0.0231 0.0145 0.0065 0.00200.986 0.964 0.989 0.864 0.987 0.957

ation T

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[15] EPA, Treability Manual, vol. 1, US Environmental Protection Agency, Washing-ton, DC, 1988, pp. 4.11–4.12.

M. Isik / Separation and Purific

Fig. 5. Lineweaver–Burk plots to determine kinetic constants, KI .

inhibition plots is KS/Rmax, while the slope of plots containing highNi(II) concentration is KS/Rmax(1 + IN/KI). The intercepts of the noinhibition plots is 1/Rmax, while the intercepts of plots containinghigh Ni(II) concentration is 1/Rmax (1 + IN/KI) as described above.As shown in Fig. 5, approximately Rmax values decreased and KSvalues increased while Ni(II) concentrations increased. Althoughthis seems non-competitive inhibition, data shows mainly mixedinhibition of substrate removal rate. Non-competitive inhibitor cancombine with either the free enzyme or the enzyme–substrate(ES) complex, interfering with the action of both. Non-competitiveinhibitors bind to a site on the enzyme other than the activesite, often to deform the enzyme, so that it does not form theES complex at its normal rate and, once formed, the ES com-plex does not decompose at the normal rate to yield products[13].

Two type of KI values were obtained for KS and Rmax becausenot only the plots differ in slope but they also do not share thesame intercept on the 1/R axis. However, as KI values are not con-stant for both KS and Rmax, the data does not completely fit intothe non-competitive model. KI values decreased either from 842 to120 mg L−1 for KS or from 725 to 596 mg L−1 for Rmax with increas-ing Ni(II) concentration from 128 to 512 mg L−1. This case showsthat the Ni(II) inhibition increases gradually with higher Ni(II) con-

centrations.

4. Conclusions

In this study, the synthetic wastewater containing Ca2+, ureaand Ni(II) was incubated with ureolytic mixed culture under batchconditions in order to determine Ni(II) inhibition to BOD degra-dation and biocalcification process. Based on the results of thisinvestigation, the following conclusions were drawn:

• The kinetics of substrate removal rate fitted to zero-order modelin the beginning of the incubation period, and to first-order modelthereafter for all the samples.

• Kinetic data clearly showed that Ni(II) concentrations, higherthan 128 mg L−1 inhibited the substrate removal according tomixed inhibition model.

• Biocatalytic calcification process was also inhibited at higherNi(II) concentrations such as 128, 256 and 512 mg L−1.

• In the calcium removal from industrial calcium-rich wastewaterNi(II) toxicity should be considered, particularly, at higher Ni(II)concentrations.

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echnology 62 (2008) 337–341 341

Acknowledgments

The Turkish Scientific and Technical Research Council (TUBITAK)and Turkish Government Planning Institution (DPT) funded thisstudy. The author would like to thank them for the financial sup-port given to the project with grant number CAYDAG 105Y262 and2006K120880-1.

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