LEAD INHIBITION ON UREA HYDROLYZING MICROORGANISMS …

2085Biotechnol. & Biotechnol. eq. 24/2010/4

Article DOi: 10.2478/v10133-010-0079-2 A&eB

Biotechnol. & Biotechnol. eq. 2010, 24(4), 2085-2091Keywords: inhibition, lead, non-competitive, ureolytic microbial activity, calcium precipitation

Introductionthe excess amount of heavy metals interferes with many beneficial uses of the water because of toxicity (4). Heavy metals such as copper, zinc, lead, mercury, chromium, cadmium, iron, nickel and cobalt are main toxic pollutants in industrial wastewaters (24). Various industrial sectors generate effluents containing high levels of heavy metals. These include liquid discharges from the mining, smelting, semiconductor, metallurgical and electroplating industry (18). heavy metals can be stimulatory, inhibitory, or even toxic in biochemical reactions depending on their concentrations. A trace level of many metals is required for activation or function of many enzymes and co-enzymes. excessive amounts, however, can lead to inhibition or toxicity. this is mostly due to the chemical binding of heavy metals to the enzymes, resulting in the disruption of enzyme structure and activities (21). A number of methods have been proposed for measuring metal toxicity in biological systems, the more commonly used ones include the measurement of enzymatic activity, the measurement of respiratory rate, the influence on the micro-organism growth parameters and the use of fluorescent and bioluminescence methods (7).

lead is a highly toxic heavy metal and is widely used in industries such as, mining and smelting, petrol, acid battery manufacturing, metal plating and finishing, printing, photographic materials, explosive manufacturing, tetraethyl lead manufacturing, ceramic and glass industries for home and consumer’s products (6, 15, 27). Some industrial wastewater may contain high lead concentrations. While Pb(ii)

concentration in wastewater of storage battery manufacturing can reach 40.3-319.4 mg/l, it varies from 0.2-843 and around 1160 mg/l in wastewater from pigment manufacturing and chlorine-alkali plants, respectively (23).

Water and wastewater containing high concentration of calcium is one of the problems in the processes of water usage and water and wastewater treatments. Precipitates of calcium are associated with young landfill leachates, reverse osmosis concentrates, industrial processes such as bone processing, paper recycling, and sugar processing (28, 29) and in industrial wastewaters that contain lime used as a cheap neutralizing agent (25). Such high calcium concentrations are problematic, because they lead to clogging of pipelines, boilers and heat exchangers through scaling (as carbonate, sulfate or phosphate precipitates) (30). existing classic chemical crystallization reactors based on the addition of a base [naoh or ca(oh)2] in the presence of nucleation site (e.g. sand grains). Such reactors are, however, often expensive, complex, and sometimes give rise to highly alkaline effluent (10) which requires neutralization for biological treatment. Recently, researchers’ studied are focused to overcome the problem of high calcium in the wastewater by using several approaches. A process using fluidized sand-coated calcium carbonate was developed to remove excess calcium hardness entering the upflow anaerobic sludge blanket (UASB) reactor (28). however, occasional clogging was observed in the fluidized treatment facility because of accumulation of calcium particles. to maintain a sufficient reactive surface for crystallization some of the grown particles should be removed regularly and replaced by smaller-diameter seeding grains (9). A novel process composed of UASB and co2-stripper was suggested to treat the liner paper wastewater and to precipitate calcium in the wastewater (14,

LEAD INHIBITION ON UREA HYDROLYZING MICROORGANISMS UNDER BATCH CONDITIONS

A. Kilic, h. Kocyigitcorespondence to: Ahmet KilicDepartment of Environmental Engineering, Aksaray University, 68100 Aksaray, TURKEYe-mail: [email protected]

ABSTRACTLead, Pb(II), inhibition of microbial calcium precipitation by ureolytic microorganisms was carried out with a glucose containing mineral medium under batch conditions over an incubation period of 120 hours. Substrate removal rate fitted a zero order up to 70th hour and first order kinetic after that time for all samples containing lead concentrations of 0-64 mg/L. The increase of Pb(II) concentrations from 0 to 64 mg/L reduced constant of substrate degradation rate from 10.26 to 4.04 mg glucose/L h, and from 0.027 to 0.005 1/h for zero (k0) and first order kinetic constant (k1), respectively. The Pb(II) at concentration higher than 16 mg/L mainly inhibited both substrate removal and nitrification process. Although nitrification was inhibited at higher concentrations of lead, its inhibition caused precipitation of calcium due to high pH and alkalinity levels in the samples. As a result that a modified Monod inhibition model was applied to BOD data, BOD removal rate inhibited non-competitively. The non-competitive inhibition constants (KI) were 10.4 mg/L for KS, and 37.3 mg/L for Rmax at Pb(II) concentration of 64 mg/L.

2086 Biotechnol. & Biotechnol. eq. 24/2010/4

19). the success and effectiveness of this process relies on the addition of naoh in the co2 stripper.

Biological approaches were also reported by a research group who proposed microbial carbonate precipitation (McP) process based on microbial urea hydrolization for calcium removal from industrial wastewater (10, 11, 12, 13). Microorganisms have long been known to catalyze the precipitation of caco3 in natural environments such as oceans, soils and saline lakes, in a process referred to as McP (3, 5). the generally accepted mechanism of McP is related to the increase of ph and dissolved inorganic carbon (Dic) of a given environment through normal physiological activities. Under aerobic conditions, one of the known McP processes is heterotrophic microbial urea hydrolysis process, in which one mole of urea is hydrolyzed by the urease enzyme to two moles of ammonium and one mole of carbon dioxide. these products can subsequently react to form ammonium and carbonate ions, which, in the presence of soluble calcium ions, can react and precipitate as caco3. Urea hydrolysis provides simultaneously a ph and co2 increase, both of which are responsible for caco3 production. in addition to these factors, precipitation process of caco3 needs nucleation site e.g. sand, suspended solids, bacteria in medium.

calcium removal from industrial wastewater is a recently new process. its environmental and operational parameters have not been extensively studied yet. in this study it was aimed to determine the inhibition of Pb(ii) to urolytic microorganisms. As it is known that metal is non competitive inhibitor to substrate removal, Pb(ii) inhibition in an urea hydrolyzing culture was investigated and evaluated for batch conditions.

Inhibition modeltraditionally a Monod type rate model [equation 1] has been widely used for describing the rate of degradation of a substrate by living cells. in other words, the substrate removal rate is commonly expressed by a deterministic model developed by Monod in 1949 (8):

[1]where S is initial substrate concentration (mg/l), R and Rmax are substrate (glucose-coD) utilization rate (mg/l h) and maximum substrate removal rate (mg/l h), respectively. kmax (µmax/Y) is maximum specific substrate utilization rate (1/h), is average biomass concentration during incubation (mg/l). µmax and Y are maximum specific growth rate (1/h) and yield coefficient (g VSS/g COD), respectively. KS is half saturation concentration (mg/l) and t is time (h).

Inhibition models are classified according to the effect of toxic compounds on the reaction rate (Rmax) and half saturation constant (KS). in the presence of increasing concentrations of metal, the impact of metals is explained by the modified Monod equations. Generally, the effect of metals is related to Rmax and KS values. As reported by few researchers, it is known

that metals inhibit noncompetitively the rate of substrate removal. Depending on the type of metal and its concentration the variations in Rmax, KS values and the inhibitions were expressed by the following equations.

[2](non-competitive inhibition model)where IL is inhibitor (lead; heavy metal) concentration (mg/l), KIL is constant of inhibition (mg/l). if equation 1 is linearized (lineweaver-Burk plot), in other words when 1/R is plotted against 1/S, a straight line is obtained (20). this line will have a slope of KS/Rmax, an intercept of 1/Rmax on the 1/R axis, and an intercept of –1/KS on the 1/S axis. Such a double reciprocal plot has the advantage of allowing much more accurate determination of Rmax and S. the double reciprocal plot can also give valuable information on inhibition. non-competitive inhibition of increasing metal concentrations to the slope and to the intercepts and the type of inhibitions is given according to equation 3 and equation 4.

slope intercept on ordinate

no metal [3]

non-competitive [4]

The rate of degradation of substrate is first order with respect to its concentration S, at high concentrations (KS >> S):

[5]Substrate removal rate is constant regardless of the substrate

concentration if S << KS. in this situation the reaction order is zero (equation 6):

[6]where k0 (mg/l h) and k1 (1/h) are zero and first-order rate constant through substrate removal, respectively.

Material and MethodsSynthetic wastewaterThe synthetic wastewater was prepared as modification of simulating medium strength municipal wastewater proposed by holakoo et al (16). it was prepared to simulate high strength municipal wastewater in the following mineral medium in mg/l: glucose-coD (1000); urea (600); cacl2 (33); MgSo4.7h2o (206); Kh2Po4 (62); Fecl3.6h2o (39); cuSo4.5h2o (0.42); MnSo4.h2o (0.25); Zncl2 (0.46), cocl2.6h2o (0.81); na2co3 (858) and nahco3 (336). Sulphuric acid was used to maintain a ph of 7.00±0.10. composition of synthetic wastewater


resulted in coD/n/P of 100/47/2.3 ratio. Urea was used in excessive concentration in the medium to serve McP process. cacl2 were also added to medium as total ca2+ of 400 mg/l during batch experiments.

Sludge production and experimental set upFlasks, of which effective volume is 150 mL, were filled with the synthetic wastewater and mineral medium described above, and 5 ml of seed activated sludge that was obtained from model continuous Stirred tank Reactor treating the domestic wastewater of Aksaray city in turkey. Flasks were shaken at 150 rpm and 30°c for 24 hours. thereafter, solids were allowed to sedimentation for 20 min, after which the supernatant was removed and analyzed for ph and nh4

+. the sludge was retained in the flasks and mineral medium, urea and glucose COD were again added to a final volume of 150 mL. This cycle was repeated for several days until a sufficient sludge was obtained. the sludge retention time (SRt) and biomass concentration in this reactor were approximately 10 days and 2000 mg/l as mixed liquor volatile suspended solids (MlVSS), respectively. Dissolved oxygen was measured above 2 mg/l during sludge production. in the experiments, the culture of bacteria was concentrated by centrifugation (5000 rpm- 2375 RcF, 5 min) and re-suspended in distilled water. the concentrated cell suspension was used to inoculate each experimental task to give a target biomass concentration. Sludge produced had a VSS/SS ratio of 0.82 and sludge volume index (SVi) of 92 ml/g.

the experiments, performed in duplicate, were carried out to determine the Pb(ii) inhibition to urea hydrolyzing bacteria, and change of other parameters such as BoD during the period, ammonium, nitrate, alkalinity, ph, SS, and VSS after experimental period of 120 hours. eighteen bottles were used, two for each of the 9 tested Pb(ii) concentrations and samples taken from them were analyzed. the composition and conditions of the medium was tabulated in Table 1. experiments were carried out in an incubator at 20°c with magnetic stirrer mixing.

TABLE 1experiment set up composition and conditions

Composition/Conditions Valuesexperiment periods (hour) 120

effective volume (ml) 97Sludge concentration (mg VSS/l) 200

coD (mg/l) 1000Urea (mg/l; 10 mM) 600.6ca(ii) (mg/l; 10 mM) 400.8

Mineral medium as described in textPb(ii) (mg/l) 0-0.5-1-2-4-8-16-32-64

initial ph 7.00±0.1

Respiration-inhibition testRespiration-inhibition tests which were based on BoD measurements were carried out into bottles of WtW oxi top system. the synthetic wastewater containing the mineral medium mentioned above was added to bottles. Pb(ii) with concentrations varying between 0.5 and 64 mg/l (a possible concentration range that is detected in wastewaters) were adjusted in the bottles by stock solution of 50 and 800 mg Pbno3-Pb/l. Duplicate controls were performed in the bottles containing no lead. oxygen consumption was monitored at specified times and compared to the control samples. Inhibition was defined as a decrease in oxygen consumption compared to the control samples.

Analytical methodsSamples were withdrawn from the mixed liquor medium after incubation time, and were centrifuged at 5000 rpm (2375 RcF) for 10 min to remove suspended solids from the medium. clear supernatants were analyzed for coD, ammonium, nitrate, and ca(ii) concentrations of samples. Standard kits (Merck-Spectroquant) and spectrometric methods were used for ammonium and nitrate. VSS, SS, SVi and ca(ii) were analyzed as specified in Standard Methods (1). pH and DO were measured by using apparatuses with the relevant probes (WtW, Germany).

Results and DiscussionThe performance of batch reactorsBoD values are shown in Fig. 1. For samples containing 0.5 and 64 mg Pb(ii)/ l, the exerted BoD values increased from 0 mg/l to 830 and 290, respectively, up to the incubation period of 70 hours. the exerted BoD values linearly decreased up to this time at all samples with higher Pb(ii) concentrations. After that time, as substrate was almost consumed by ureolytic bacteria, the rate of substrate consumption gradually decreased with respect to Monod kinetic. BoD data were clearly shown that Pb(ii) concentrations from higher than 16 mg/l inhibited the activities of ureolytic bacteria. the response of bacteria to higher Pb(ii) concentration was similar as shown in Fig. 1.

0

200

400

600

800

1000

1200

0 20 40 60 80 100 120 140

I n c u b a t i o n T i m e ( hour )

B O

D (

mg

/ L

)

0 0,5 1 2 4 8 16 32 64

Fig. 1. exerted BoD values of samples containing various Pb(ii) concentrations (mg/l) during the incubation


Calcium removalMcP process is based on microbial urea hydrolysis, thus the increase of the ph and ammonium values results in the precipitation of soluble calcium in the medium. As shown in Fig. 2 calcium has been removed at samples containing Pb(ii) concentrations higher than 16 mg/l. Soluble calcium values decreased from 400 mg/l to 108 mg/l for Pb(ii) concentration of 64 mg/l, while it was reduced to 336 mg/l for Pb(ii) free sample at the end of the incubation period. Also high levels of alkalinity and ph were observed at the samples where calcium was removed. high alkalinity levels and ph caused the precipitation of calcium as described by other researchers (2).

0

100

200

300

400

500

600

0 0.5 1 2 4 8 16 32 64

L e a d C o n c e n t r a t i o n ( mg / L )

A l

k a

l i n

i t y

( m

g C

aCO

3 / L

)

Ca

2+ (

mg

/ L )

6.0

7.0

8.0

9.0

pH

ca Alkalinity ph

Fig. 2. the relationship of alkalinity, ph and ca(ii) values at the samples

As substrate was entirely consumed in the samples containing lower Pb(II) concentrations nitrification could start. Data for ammonium and nitrate confirmed the nitrification process at these samples as shown at Fig. 3.

0

2

4

6

8

10

12

0 0.5 1 2 4 8 16 32 64


A m

m o

n i

u m

; N

i t r

a t

e (

mM

)

0

2

4

6

8

10

12

C a 2+ ( m

M )

nitrate Ammonium calcium

Fig. 3. the change of ammonium, nitrate and calcium values at the end of incubation

pH and alkalinity decreased due to possible nitrification. It is known that nitrification is an alkalinity consumption process that causes conversion of the ammonium to nitrate, which was measured in medium at the end of incubation period. As a consequence, the nitrification inhibits the calcification process. As nitrification both consumed dissolved inorganic carbon and decreased ph, the medium was not appropriate for the calcium precipitation. however, as the ammonium converted partly

to nitrate (nitrification), medium was maintained for calcium precipitation at high Pb(ii) concentrations. it is possible that high Pb(II) concentrations also inhibited the nitrification process in the medium because of low growth rates of nitrifying bacteria and their extremely high sensitivity to toxic metals (26).

Alkalinity increased up to 543 mg caco3/l at the sample containing Pb(ii) concentration of 16 mg/l, but decreased to 203 mg caco3/l at the lead-free sample because of consumption of in the medium. the ph increased as the production of ammonia from urea increased, but did not exceed 8.24, as a result of the ammonium buffer equilibrium. this ph value, which enables optimum microbial growth, is one of the main advantages of biocatalytic calcification process compared to chemical ones.

the low ratio of the VSS/SS in the sample containing higher Pb(ii) concentrations also indicated the precipitation of ca(ii) together with bacteria as shown in Fig. 4. Decreasing of the VSS/SS ratio mainly resulted from the presence of caco3, which was measured as SS. VSS/SS ratio decreased from 0.68 for Pb(ii) free sample to 0.383 for the concentrations of 16 mg/l.

0

200

400

600

800

1000

1200

0 0.5 1 2 4 8 16 32 64


S S

; V S

S (

mg

/ L )

0

0.2

0.4

0.6

0.8

1

V S S / S S

SS VSS VSS/SS

Fig. 4. the changes of VSS, SS and VSS/VS of the samples

Determination of kinetic coefficientsin order to determine the type of inhibition the half velocity constants (KS) and maximum substrate removal rates (Rmax) were calculated. the substrate was removed according to the zero order at low substrate concentrations and first order kinetic reaction at high substrate concentrations according to Monod equation. considering this and taking into account the zero and first order substrate removal kinetic, the zero and first order reaction rate during incubation period were equalized to the reaction rate of the Monod kinetic to determine the KS values and Rmax.

Since the Monod kinetic could not be applied (Monod kinetics shows very low correlation, data not shown) to determine the substrate removal rates and the kinetic coefficients through biodegradation course the reaction rate of the approaches given in equation 5 and equation 6 were used to determine the kinetic constants.


Rmax values were calculated from the substrate removal rate up to 70th hour. in this period, substrate removal rate was appropriate to zero order kinetic equation. After 70th hour, substrate removal rate was first order for all samples containing metal in concentrations between 0 and 64 mg/l. Data shows high correlation with corresponding kinetics on the graph in Fig. 5a. For up to 70th hour, reaction rate fit to zero order, but after that time, to first order.

Fig. 5 depicts zero and first order plots in order to determine the kinetic constants for samples containing various Pb(ii) concentration. The kinetic and correlation coefficients relevant to the zero and first order rate are summarized in Table 2. the comparison of regression coefficient (r2) relevant to zero and first order rate constants (k0 and k1) showed that substrate was removed according to the zero order reaction kinetic up to 70th hour, and first order reaction kinetic after this period. increase in Pb(ii) concentration from 0 to 64 mg/l reduced the k0 values from 10.26 to 4.04 mg/l h, k1 values- 0.027 and 0.005 1/h in batch studies. Decrease in these values clearly showed that upper 16 mg/l of Pb(ii) concentrations inhibited the substrate utilization rate under this experimental condition.

Researchers (22) reported that lead produced inhibition of 67% on specific oxygen uptake rate (sOUR) for activated sludge culture and ammonium uptake rate (AUR) for nitrifying

culture at concentration of 16.9 mg Pb(ii)/l. on the contrary, tchobanoglous et al. (26) reported that Pb(ii) of 0.1 mg/l had an inhibitory concentration threshold effect on heterotrophic organism. however, there are no data with comparable values in relation to effect of metals on ureolytic aerobic heterotrophic bacteria in literature.

For samples that varied from no metal and concentration of 64 mg/l, reactions may be equalized to the zero order substrate kinetic with respect to equation 6. After 70th hour, reaction rate may be equalized to the first order to determine KS in Monod kinetic as following the equation:

[7]where Si is substrate concentration at i step (mg/l). KS can be calculated from rearrangement of equation 7 for data after 70th hour and substituted in equation 8 reported by isik and Sponza (17). Rmax and KS values may be obtained from data as following Table 3.

[8]where n is number of data obtained during the time course of batch study.

TABLE 2the kinetic constants obtained in aerobic batch tests during BoD degradation

Constant Period(hour)

Pb(II) Concentration (mg/L)0 0.5 1 2 4 8 16 32 64

k0 (mg/l h) up to 70(0. order)

10.266 11.600 10.932 11.001 10.666 11.418 5.033 4.493 4.040r2 0.992 0.993 0.992 0.986 0.992 0.993 0.971 0.969 0.966

k1 (1/h) after 70(1. order)

0.027 0.065 0.044 0.077 0.021 0.027 0.006 0.005 0.005r2 0.978 0.951 0.938 0.989 0.982 0.950 0.976 0.953 0.985

0

200

400

600

800

1000

1200

0 20 40 60 80I n c u b a t i o n T i m e ( hour )

S i

0 0.5 1 2 4 8 16 32 64

(a)

0

2

4

6

8

70 80 90 100 110 120 130I n c u b a t i o n T i m e ( hour )

Ln S

i

0 0.5 1 2 4 8 16 32 64

(b)

Fig. 5. (a) Zero order kinetic up to 70th hour of incubation period; (b) first order reaction kinetic after 70th hour for batch reactor


TABLE 3Maximum substrate utilization rate, half saturation concentration, and the values of inhibition constant

ConstantLead Concentration (mg/L)0 – 8 16 32 64

Rmax (mg/l h) 11.0 ± 0.5 5.0 4.5 4.0KS (mg/l) 306.5 ± 143.4 872.1 890.2 807.5

Kil for KS (mg/l) not applicable 3.1 5.2 10.4Kil for Rmax (mg/l) not applicable 13.5 22.2 37.3

±shows standard deviation

-0.5

0.5

1.5

2.5

3.5

4.5

-0.003 0.002 0.007 0.012 0.017 0.022

1 / S

1 / R

no inhibition 16 32 64

I

Fig. 6. lineweaver – Burk plots according to kinetic constants

Fig. 6 clearly shows the inhibition of increasing metal concentrations accomplished with the inhibition of substrate degradation. The reciprocal fits (Lineweaver-Burk plots) of inhibition given in equation 4 showed that Pb(ii) caused non-competitive inhibition on BoD degradation resulting in increases in KS values and decreasing Rmax. the KS and Rmax values, in the aforementioned equations, were calculated using equation 6 and equation 8 that provide a mean to determine the inhibition constants Kil. the slope of the no inhibition plots is (KS/Rmax) while the slope of plots containing high metal concentration is (KS/Rmax).(1+il/Kil). the intercepts of the no inhibition plots is (1/Rmax), while the intercepts of plots containing high metal concentration is (1/Rmax)(1+il/Kil) as shown in equation 3 and equation 4. As data obtained from batch conditions and values in Table 2 showed no inhibition on BoD removal rate, KS and Rmax values were assumed to be the approximately the same, hence, the average values were calculated for data on which no inhibition was observed for metal concentrations up to 16 mg/l. As a result of this approach, the obtained kinetic values are tabulated in Table 3 for samples with no inhibition and inhibition.

As shown in Fig. 6 in the non-competitive inhibition, the Rmax decreased and KS values increased while Pb(ii) concentrations increased. non-competitive inhibitors can combine with either the free enzyme or the enzyme-substrate (eS) complex, interfering with the action of both. non competitive inhibitors bind to a site on the enzyme other than the active site, which often leads to conformation changes of the enzyme, so that

it does not form the eS complex at its normal rate and, once formed, the eS complex does not decompose at the normal rate to yield products (20).

two type of Kil values were obtained for KS and Rmax because not only the plots differed in slop but also did not share the same intercept on the 1/R axis. however, as Kil values were not constant for both KS and Rmax, non-competitive model completely did not fit for these data. Kil values increased either from 3.1 to 10.4 mg/l for KS or from 13.5 to 37.3 mg/l for Rmax as Pb(ii) concentration increased from 16 to 64 mg/l. this case shows that the increase of metal inhibition strength decreases gradually with an increase of metal concentration.

ConclusionsThe kinetic of substrate removal rate fitted to zero order up to 70th hours of the incubation period, but to first order model after that time for all the samples. Kinetic data clearly showed that Pb(ii) concentration higher than 16 mg/l inhibited non competitively the substrate removal. Surprisingly, ca(ii) could be significantly removed in the samples containing lead concentration higher than 16 mg/L as nitrification was inhibited at these samples. As nitrification is an alkalinity consumption process, calcification was mainly observed for non nitrified samples. Therefore nitrification and Pb(II) toxicity should be considered in the calcium removal from industrial calcium-rich wastewater.

REFERENCES1. Andrew E. (2005) Standard Methods for the examination

of Water and Wastewater, American Public health Association, Washington Dc, USA.

2. Blum D.J.W. and Speece R.E. (1991) J. Water Pollut. control Fed., 63, 198-207.

3. Castanier S., Metayer-Levrel G.L., Perthuisot J.P. (1999) Sediment. Geol., 126, 9-23.

4. Chang W.C., Hsu G.S., Chiang S.M., Su M.C. (2006) Bioresource tech., 97, 1503-1508.

5. Douglas S. and Beveridge T.J. (1998) FeMS Microbiol. ecol., 26, 79-88.

6. Gercel O. and Gercel H.F. (2007) chem. eng. J., 132, 289-297.

7. Gikas P. (2007) J. hazard. Mater., 143, 246-256.8. Grady Jr.C.P.L., Daigger G.T., Lim H.C. (1999)

Biological wastewater treatment, Marcell Dekker inc., new York, p. 261.

9. Graveland A., Dijk J.C., Moel P.J., Oomen J.H.C.M. (1983) J. Am. Water Works Ass., 75, 619-625.

10. Hammes F., Boon N., Clement G., de Villiers J., Siciliano S.D., Verstraete W. (2003) Appl. Microbiol. Biotechnol., 62, 191-201.


11. Hammes F., Boon N., de Villiers J., Verstraete W., Siciliano S.D. (2003) Appl. environ. Microbiol., 69, 4901-4909.

12. Hammes F., Seka A., Hege K.V., Wiele T.V., Vanderdeelen J., Siciliano S.D., Verstraete W. (2003) J. chem. technol. Biotechnol., 78, 670-677.

13. Hammes F., Seka A., Knijf S., Verstraete W. (2003) Water Res., 37, 699-704.

14. Han K.C. and Lee W.K. (2003) Process Biochem., 38, 925-931.

15. Helal N.H., El Rabiee M.M., Abd El Hafez Gh.M., Badawy W.A. (2008) J. Alloy. comp., 456(1-2), 372-378.

16. Holakoo L., Nakhla G., Bassi A.S., Yanful E.K. (2007) chemosphere, 66, 849-857.

17. Isik M. and Sponza D.T. (2005) Process Biochemistry, 40, 1053-1062.

18. Karri S., Sierra-Alvarez R., Field J.A. (2006) chemosphere, 62, 121-127.

19. Kim Y.H., Yeom S.H., Ryu J.Y., Song B.K. (2004) Process Biochem., 39, 1393-1399.

20. Lehninger A.L. (1977) the molecular basis of cell structure and function biochemistry, Worth Publishers, inc., new York, p. 200.

21. Li C. and Fang H.H.P. (2007) chemosphere, 67, 668-673.

22. Madoni P., Davoli D., Guglielmi L. (1999) Water Res., 33, 2459-2464.

23. Patterson J.W. (1985) industrial Wastewater treatment technology, Butterworth Publishers, Boston.

24. Sarioglu M., Atay U.A., Cebeci Y. (2005) Desalination, 181, 303-311.

25. Svardal K. and Kroiss H. (1988) Fifth international symposium on anaerobic digestion, Bologna, italy, p. 335-340.

26. Tchobanoglous G., Burton F.L., Stensel H.D. (2003) Wastewater engineering: treatment and reuse, Metcalf & eddy inc., new York.

27. Tran T.H.H., Warounsak L., Ajit P.A. (2007) Bioresource technol., 98, 2538-2548.

28. Van Langerak E.P.A., Hamelers H.V.M., Lettinga G. (1997) Water Sci. tech., 36, 341-348.

29. Van Langerak E.P.A., Gonzales-gill G., Van Aelst A., Van Lier, J.B., Hamelers H.V.M., Lettinga G. (1998) Wat. Res., 32, 1255-1263.

30. Yu H.Q., Tay J.H., Fang H.H. (2001) Water Res., 35, 1052-1060.

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LEAD INHIBITION ON UREA HYDROLYZING MICROORGANISMS …