Journal of Industrial and Engineering Chemistry 18 (2012) 1908–1911

Effect of urea concentration on microbial Ca precipitation

Mustafa Is ık a,*, Levent Altas a, Samet Ozcan a, Ismail Sims ek a, Osman Nuri Agdag b, Ali Alas c

a Aksaray University, Engineering Faculty, Environmental Engineering Department, Aksaray, Turkeyb Pamukkale University, Engineering Faculty, Environmental Engineering Department, Denizli, Turkeyc Necmettin Erbakan University, A.K. Education Faculty, Biology Department, Konya, Turkey

A R T I C L E I N F O

Article history:

Received 12 December 2011

Accepted 6 May 2012

Available online 23 May 2012

Keywords:

Urea

Calcification

Precipitation

Calcium

Wastewater

A B S T R A C T

High calcium concentrations are problematic, because they lead to clogging of pipelines, boilers and heat

exchangers through scaling, or malfunctioning of aerobic and anaerobic reactors. Urea hydrolysis

provides simultaneously a pH and CO2 increase, both of which are responsible of CaCO3 production. This

study was carried out to determine urea concentrations between 5 and 20 mM on ureolytic mixed culture

treating synthetic wastewater. The optimum urea concentration was found as 15 mM for effective

calcium removal. This work showed the feasibility of urea-based microbial carbonate precipitation as an

alternative Ca2+ removal technology.

� 2012 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights

reserved.

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Journal of Industrial and Engineering Chemistry

jou r n al h o mep ag e: w ww .e lsev ier . co m / loc ate / j iec

1. Introduction

Calcium-rich effluents are associated with landfill leachates,reverse osmosis concentrates, and industrial processes such asbone processing and paper recycling. The paper recycling processrequires large volumes of water (up to 1.4 billion m3 per year forEurope), while producing effluents containing 10–40 mM Ca2+

[1,2]. Such high calcium concentrations are problematic, becausethey lead to clogging of pipelines, boilers and heat exchangersthrough scaling (as carbonate, sulfate or phosphate precipitates),or malfunctioning of aerobic and anaerobic reactors [3–5]. As aremedy to this problem, the industry typically uses chemicalcrystallization reactors which increase pH via the addition of abase, e.g., Ca(OH)2 or NaOH, and thereby precipitate CaCO3 in thepresence of crystal nucleation sites, e.g., sand grains. These reactorsare efficient but often require complex monitoring and control and,as a drawback, can give rise to highly alkaline effluents [6].

Biological mineral precipitation represents an interestingalternative means for the treatment of industrial wastewaters aswell as groundwater contaminated with excessive amounts of‘‘unusual’’ pollutants such as heavy metals, radionuclides, phos-phate, and salts [7]. Four equilibrium relationships are potentiallyneeded to describe carbonate system, which are presented here(ignoring activity corrections) [8]. In open (i.e., open to theatmosphere) systems, there is an interchange between atmospher-

* Corresponding author. Tel.: +90 382 2882315; fax: +90 382 2882298.

E-mail addresses: mustafaisik@aksaray.edu.tr, mustafaisik55@hotmail.com

(M. Is ık).

1226-086X/$ – see front matter � 2012 The Korean Society of Industrial and Engineer

http://dx.doi.org/10.1016/j.jiec.2012.05.002

ic CO2 and H2CO3* in solution, with the gas–water equilibriumdescribed by Henry’s law as follow:

H2CO3�ðaqÞ $ CO2ðgÞ þ H2O KH ¼

½PCO2�

½H2CO3��

¼ 31:6 atm=M at 25 �C (1)

where, by convention, ½H2CO3�� equals the sum of the carbonic acid

concentration, [H2CO3], plus the dissolved carbon dioxide concen-tration, [CO2(aq)], and KH is the Henrys law constant. In water,carbonic acid is diprotic (i.e., it dissociates in two steps). First, itdissociates to bicarbonate, HCO3

�, and a proton,

H2CO3� $ Hþ þ HCO3

� Ka;1 ¼½Hþ�½HCO3

��½H2CO3

��¼ 4:3 � 10�7 mol=L at 25 �C (2)

where Ka,1 is the equilibrium constant. Second, bicarbonate iondissociates to carbonate ion, CO3

2�, and a proton,

HCO3$ Hþ þ CO32� Ka;2 ¼

½Hþ�½CO32��

½HCO3��

¼ 4:7 � 10�11 mol=L at 25 �C (3)

where Ka,2 is the equilibrium constant. Based on these equilibria, amass balance on the total concentration of dissolved inorganiccarbon (DIC) species gives

CT ¼ ½H2CO3�� þ ½HCO3

�� þ ½CO32�� (4)

In open systems, ½H2CO3�� is constant with pH (Eq. (1)), while CT

changes with the pH. For example, as the pH increases, ½HCO3�� and

ing Chemistry. Published by Elsevier B.V. All rights reserved.

M. Is ık et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 1908–1911 1909

½CO32�� increase [Eqs. (2) and (3)], causing CT to increase. However,

in closed systems (i.e., closed to the atmosphere), CT is constantwith pH if no precipitation occurs. If a carbonate solid is alsopresent or forms, an additional reaction describing its solubility isrequired. For example, for calcium carbonate, CaCO3(s),

CaCO3ðsÞ $ Ca2þ þ CO32� KSP ¼ ½Ca2þ�½CO3

2��

¼ 5 � 10�9 mol2=L2 at 25 �C (5)

where KSP is the solubility product. The product of the calcium ionand carbonate concentrations must exceed the solubility productfor precipitation to occur (i.e., the solution must be oversaturated).This can be quantified using the saturation state, S, of the solution:

S ¼ ½Ca2þ�½CO32��

KSP(6)

when S > 1, the reacting solution is over- or supersaturated.A key process control for promoting biocementation via

carbonate precipitation is to supply substrates whose transforma-tion shifts the carbonate system to increase the carbonateconcentration while ensuring adequate concentrations of appro-priate cations, such as calcium, to promote precipitation [Eq. (5)].

The precipitation of CaCO3 and other insoluble carbonates canbe induced via a variety of heterotrophic and autotrophic microbialprocesses. Aerobic and anaerobic oxidation of organic compoundsresulting in the addition of dissolved H2CO3

� will increase the totalconcentration of the dissolved carbonic species [Eq. (4)]. If suchoxidations occur in a well-buffered neutral or alkaline environ-ment, at least some of the H2CO3

� produced will be transformedinto carbonate [Eqs. (2) and (3)], which will precipitate if sufficientCa2+ or other appropriate cations are present [Eq. (5)] [9]. Forexample, Krumbein [10] demonstrated the formation of aragoniteand other calcium carbonates under these conditions by hetero-trophic bacteria and fungi in media containing organic matter. If anenvironment is not already well buffered and neutral or alkaline,an increase in alkalinity and pH is necessary to ensure that the CO2

produced is being transformed into carbonate [Eqs. (2) and (3)] forforming carbonate precipitation. Alkalinity is a measure of thecapacity of water to neutralize strong acid. In natural waters, this ismostly attributable to the bases of the carbonate system (HCO3

�,CO3

2�) and OH–; therefore, total alkalinity (TA) is often defined as;

TA ¼ ½HCO3�� þ 2½CO3� þ ½OH�� � ½Hþ� (7)

However, in some natural and contaminated waters, other saltsof weak acids such as phosphates and weak organic acids maycontribute to the TA. Note that precipitation of calcium carbonate[Eq. (5)] will decrease the pH and TA [Eqs. (2), (3), (5) and (7)]. Anincrease in alkalinity and/or pH can be accomplished directly viaseveral microbial mediated processes. One of these processes is theaerobic or anaerobic biotransformation of organic nitrogencompounds, either via oxidation (e.g., of amines, amino acids,purines, pyrimidines), or hydrolysis (e.g., of urea) [9]. In particular,much of the interest in promoting biocalcification in soils andother construction materials has focused on urea hydrolysis (orureolysis) [11].

COðNHÞ2 þ Hþ þ 2H2O ! 2NH4þ þ HCO3

� (8)

Ca2þ þ 2HCO3�$ CaCO3ðsÞ þ CO2 þ H2O (9)

Biotransformation of organic nitrogen compounds has theadvantage of releasing NH4

+ as well as CO2, resulting in an increasein pH and alkalinity [Eq. (8)]. This creates an environment where atleast some of the CO2 produced will be transformed into carbonate.Thus, in unbuffered environments containing adequate amounts ofCa2+ and other cations, CaCO3 precipitation can result if the

solubility product is exceeded [Eqs. (6) and (9)]. For example,Stocks-Fischer et al. [12] observed urea degradation in batchsystems to increase ammonia and pH levels, with microbiologicalCaCO3 precipitation beginning at pH 8.3, completed at pH 9.Similar trends were observed by others [13–17].

In a previous study [16], we have reported on the effect ofhydraulic retention times of biocatalytic calcium reactor perfor-mances for calcium removal from synthetic wastewater repre-senting paper mill effluent and domestic wastewater. Ureaconcentration effect on a microbial carbonate precipitation(MCP) process, based on urea hydrolysis, was investigated as apotential alternative to chemical carbonate precipitation. Ureadegradation is a simplistic process, and since it is based on theworking of the urease enzyme, which is common in manyorganisms, it can easily be integrated in an existing biologicalwastewater treatment system. Moreover, urea is a relativelyinexpensive chemical and provides upon hydrolysis simultaneous-ly a pH increase and DIC increase. The aim of this work was firstlyto determine the effect of urea concentration on BCR reactorperformance in terms of the organic matter removal over time, andsecondly to evaluate the ammonia toxicity on ureolytic mixedculture.

2. Materials and methods

2.1. Wastewater characteristics and BCR for continuous feeding

studies

The content of the intended synthetic wastewater that wasused for continuous feeding studies was prepared by composingdomestic wastewater with average degree of pollutants, as advisedby Holakoo et al. [18], and wastewater of a paper factory, which thecontents of the water is advised by Kim et al. [19]. The content ofthe prepared synthetic composition that was used in the studies isgiven in Table 1. The aim of mixing wastewater composition withthe domestic wastewater composition was the combined removalof domestic wastewater caused by the personnel in the paperfactories and therefore to prevent the absence of major or minorfeeding element in paper factory wastewater. As the first step,wastewater simulation with no containment of Ca, as seen in Table1, was used for the production of sufficient biomass of ureahydrolysis and The COD/N/P ratio of wastewater was elicited as100/28/1. After that, 601.2 mg/L of Ca2+ concentration was addedto synthetic wastewater in order to simulate Ca-rich wastewatersuch as paper factory. Only, urea was provided to syntheticwastewater for promoting of Ca removal from combined syntheticdomestic and paper factory wastewater.

BCR had dimensions of 7 cm � 18 cm � 30 cm; and consisted ofaeration basin of 1.5 L and sedimentation basin of 0.65 L. Aerationbasin and sedimentation unit was separated by a perforated platethat allowed water transition (Fig. 1). The transition of precipitatedsludge to aeration unit provided the sludge recycling ratio to befixed to 1 by the help of a 2–3 cm opening in the bottom of theplate. BCR, with the assumption that there was no biomass ineffluent, was operated by using sludge retention time (SRT) of5 days with sludge wasted from aeration tank. Actual SRTs given inresults sections were calculated with taking into consideration ofmeasured effluent biomass. The composition in the reactor wascreated by using air pump that could provide sufficient oxygen(>2 mg/L) in the aeration basin. Simulated wastewater was givento the reactors, from the condition of 4 8C, by peristaltic pumps.Reactor and removal studies were implemented in a fixedtemperature of 25 8C. Reactor was operated for 111 days athydraulic retention times of 8.06 � 0.87 h, after it reached stableconditions, by changing the urea concentration about 20 days.

Table 1Characteristics of simulated wastewater.

The functions of material used Material used Conc., mg/L Target conc., mg/L

Paper and domestic wastewater C6H12O6�H2O 1548 1500 mg glucose–COD/L

Paper and domestic wastewater CaCl2 1665 601.2 mg Ca/L

Paper and domestic wastewater and BCR process CO(NH2)2(urea) 0–300–601–901–1202 mg/L 0–140–280–420–560 mg N/L

Paper and domestic wastewater MgSO4�7H2O 1540 600 mg SO4/L

Paper and domestic wastewater Na2CO3 477 270 mg CaCO3/L

Paper and domestic wastewater NaHCO3 378 270 mg CaCO3/L

Domestic wastewater and trace element KH2PO4 66 15 mg P/L

Domestic wastewater FeCl2�4H2O 19 4 mg Fe/L

Domestic wastewater CoCl2�6H2O 0.404 0.10 mg Co/L

Domestic wastewater ZnCl2 0.229 0.11 mg Zn/L

Domestic wastewater NaMoO4�2H2O 0.208 0.08 mg Mo/L

Domestic wastewater CuSO4�5H2O 0.118 0.03 mg Cu/L

Domestic wastewater MnSO4�H2O 0.123 0.04 mg Mn/L

M. Is ık et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 1908–19111910

2.2. Analytical methods

Total suspended solid (TSS) and volatile suspended solid (VSS)of samples were measured by the filtration technique usingmembrane filters with pore sized 0.45 mm [20]. CODs in influentand effluent were determined colorimetrically using a UV–VISSpectrofotometer (Spectro UV–VIS RS, USA) by closed refluxcolorimetric methods and standardized procedures [20]. Samplesfrom the reactor were analyzed for alkalinity, pH, and sludgevolume index (SVI) according to Standard Methods [20]. Ammo-nium was measured photometrically a VIS spectrophotometer(ThermoSpectronic-Aquamate-USA) by Merc-Spectroquant kitsand total nitrogen (TN) measurements were realized by TOC-TN(Shimadzu TOC-VCPN) analysis system.

3. Results

Fig. 2 shows the effect of urea concentration on evolutionalkalinity, ammonium and pH in the reactor. Alkalinity wasproduced from urea hydrolysis which cause two mol ammonia andone mol CO2 from 1 mol urea and, organic matter degradation fromheterotrophic activity of ureolytic mixed culture (UMC). Ammoni-um levels were decreased with decreasing urea concentrationadded to synthetic wastewater. Generally ammonium concentra-tions at all runs were on average slightly lower than expectedtheoretically levels (10 mM urea equals to 360 mg NH4

þ). Lowammonium levels in effluent may be attributed to a combination ofvolatilization, microbial uptake of biosynthesis [21], possiblenitrification and not completely conversion of urea to ammoniadue to urease activity. Little population of nitrifiers compared toheterotrophic mixed culture may be present in reactor and mayconverted ammonium to nitrate for energy requirements. At highurea concentrations, low ammonium levels of effluent may be

Fig. 1. The schematic configuration of the continuous BCR.

attributed to insufficient urease activity of microbial population.As ammonia quantities increased with higher urea concentrations,pH increased to 8.36 from 7.5 with increasing urea concentrations.Ammonia productions from urea degradation, CO2 productionfrom urea hydrolysis and oxidation of organic matter, andcarbonate precipitation has significant influences on the eventualpH. Whereas ammonia production results in a pH increase, CaCO3

precipitation leads to acidification due to consuming of alkalinityfrom bulk water [21]. pH increase from ammonia production wasmore powerful than pH decreases from CaCO3 precipitation, as pHsincreased continuously with increasing urea concentrations in thisstudy.

Fig. 3 compares the removal efficiencies COD, calcium and TN inthe BCR reactor during experimental runs. While higher CODremoval efficiency was obtained at concentration of 5 mM urea,lower COD removal efficiencies were obtained at concentration of20 mM urea due to possible ammonia toxicity and at zero ureaconcentration due to N deficiencies for biosynthesis. Calciumremoval was increased proportionally with increasing of ureaconcentrations which cause pH and alkalinity evolution resultingwith Ca precipitation. As the primary objectives of the BCR reactorwere effective calcium removal, optimum urea concentration canbe accepted as 15 mM for 15 mM Ca in influent at these conditions.Based on Eqs. (8) and (9), theoretically, 15 mM of urea concentra-tion is required to precipitate 15 mM calcium from water. It issuggested that minimum concentration of urea should be equal tocalcium concentration needed to removal. Similar findings werefound in a study [15], which argued that a minimum of 12 mM urea(=0.72 g/L) is required to precipitate 12 mM calcium.

Significant calcium ions were removed from synthetic waste-water via CaCO3 precipitation. Fig. 4 shows lower SVI values and

Fig. 2. Alkalinity, ammonium and pH values in BCR reactor.

Fig. 4. SVI values, MLVSS/MLSS ratios and, MLSS concentrations of BCR sludge.

Fig. 3. COD, TN and, calcium removal efficiencies of BCR reactor.

M. Is ık et al. / Journal of Industrial and Engineering Chemistry 18 (2012) 1908–1911 1911

MLVSS/MLSS ratios and higher MLSS concentrations were ob-served at higher urea concentrations. While the VSS remainedapproximately constant the TSS increased in the reactor withincreasing CaCO3 crystals with at high urea concentrations.Previous studies of ureolytic MCP also described calcite formationrather than other CaCO3 polymorphs [12,22]. Lower SVI valuesshow the crystalline characteristics of the sludge allowing forextremely rapid sedimentation in the test and sedimentation partof reactor. Hence, it is said that one of the advantages of MCPprocess is increasing sludge settleability, which provides goodsolid/water separation.

In conclusion, BCR reactor combining mixed ureolytic bacteriaon calcareous sludge flocs, represent an efficient process forcalcium removal from synthetic industrial wastewater and good

sludge settleability due to heavier CaCO3 crystals. Optimum ureaconcentration for this study was found as 15 mM for 15 mM Ca ininfluent at these conditions. This work showed the feasibility ofurea-based microbial carbonate precipitation as an alternativeCa2+ removal technology.

Hydrolysis reactions of urea by ureolytic cultures treatedsynthetic wastewater resulted in precipitation of Ca. If this processcan be optimized via appropriate recovery technology of calciumproduced wastewaters, treated wastewater would be used atreusing applications of treated wastewater. Major drawbacks ofthis process are urea cost and N-pollution. However, if somewastewaters containing organic N and/or ammonia and organicmatter are present with calcium rich wastewater, this technologycould be favorable for effective Ca and organic matter removal.Also as BCR reactors can be used the treatment of waters withelevated levels of heavy metals, phosphate and radionuclides, thistechnology may be favorable in use for the removal of suchcontaminants in future.

Acknowledgement

This study was supported by the TUBITAK CAYDAG 105Y262project.

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