Introduction system [2].

1. Introduction system [2].

To avoid health hazards it is essential to remove Heavy metal pollution in the aquatic environment these toxic heavy metals from waste water before its has become of great environmental concern since they disposal. Main sources of heavy metal contamination are non-biodegradable. Metals transferred in food include urban industrial aerosols, solid wastes from chain as a result of leaching from industrial wastewater, animals, mining activities, industrial and agricultural

polluted soils and water [1].

chemicals. Heavy metals also enter the water supply Industries that work with car batteries, sheets of from industrial and consumer water or even from acid semifinished metal, additives in gasoline, ammunition rain which breaks down soils and rocks, releasing and scrap iron from car batteries are the main sources heavy metals into streams, lakes and ground water [3]. of effluents containing lead. Besides respiratory Techniques presently in existence for removal of problems, lead provokes alterations in blood and urinary heavy metals from contaminated waters include:

reverse osmosis, electrodialysis, ultrafiltration, Corresponding author: Harith Jabbar Fahad Al-Mathkhury,

ion-exchange, chemical precipitation, Ph.D., assistant professor, research fields: pathogenic bacteria,

bacterial ecology. E-mail: harithfahad@scbaghdad.com. phytoremediation, etc. However, all these methods

Bioremoval of Aquatic Environment Lead by Immobilized Cells of Enterobacter spp.

have disadvantages like incomplete metal removal,

agar and stored at 4°C.

high reagent and energy requirements, generation of

2.3 Biomass Preparation

toxic sludge or other waste products that require careful disposal [4]. With increasing environmental awareness Bacterial isolates were grown in a 1 L Erlenmeyer and legal constraints being imposed on discharge of

flask containing 0.5 L Brain heart infusion broth effluents, needs for cost-effective alternative medium (Himeida, India) at 37°C , 100 rpm for 24 h, technologies are essential.

subsequently, biomass was harvested by centrifugation

Of the different biological methods, at 6000×g for 15 min at 4°C and washed twice with bioaccumulation and biosorption have been deionized distilled water (DDW), 1 mL was taken for demonstrated to possess good potential to replace

the dray weight measurement.

conventional methods for the removal of dyes/metals

2.4 Lead Solution

[5]. It is a biological method of environmental control Lead stock solution was prepared by dissolving

and can be an alternative to conventional contaminated 1.5985 g of lead nitrate in 1 L of DDW. However,

water treatment facilities. It also offers several

working solution 100 mg/L, pH 4 was prepared from advantages over conventional treatment methods

this stock solution.

including cost effectiveness, efficiency, minimization of chemical/biological sludge, requirement of

2.5 Biosorption Protocol

additional nutrients, and regeneration of biosorbent Biosorbents (bacterial cells) were added to the lead

with possibility of metal recovery. working solution in a final concentration reached 0.5

This research aimed to study the biosorption of lead mg/L, as triplicates, for one hour at 40°C in a shaker

by bacterial cells in batch and continuous systems, incubator (2,000 rpm). Control lead solution (free of

obtaining equilibrium parameters in a static system to bacteria) was prepared as well. After that, all lead

aid in the dynamic operation of bioreactors for the treatment of ionic lead in high concentrations.

solution were centrifuged at 4°C for 30 min at 6,000×g and the lead concentration was estimated in the

2. Materials and Methods

supernatant using flame atomic absorption

2.1 Sampling spectrophotometer [7]. The amount of biosorbed lead

was taken to be the difference between the lead Twenty four samples were collected in sterile 1 L

concentration in control and lead concentration in glassware containers from the wastewater of the

supernatant. The best biosorbent was elected for further treatment unit in the general state of batteries industries

continuous flow experiments.

in Baghdad-Iraq.

2.6 Continuous Flow System

2.2 Bacterial Isolation and Identifications Fig. 1 illustrated the plant employed in this study

After good mixing, six decimal dilutions for each consists of a glassware column bioreactor (25 cm in sample were done. 0.1 mL of each dilution was

length) plugged with two rubber plugs at both ends cultured on Nutrient agar, MacConky agar (Himeida,

penetrated by glass tubes which is connected to plastic India) via spreading using L-shaped glass spreader on

tubes. At the lower end of the glassware column, the two plates per dilution. All plates were cultivated at

plastic tube is connected to a peristaltic pump that 37°C for 24 h. Identification was completed according

connected to lead solution. The upper end is connected to Bergey’s manual [6] and api 20 E system. It was

to collection vessel via plastic tube. The best maintained by monthly subculturing using nutrient

desorption solution was elected for further studies.

Bioremoval of Aquatic Environment Lead by Immobilized Cells of Enterobacter spp.

homogeneity was reached. E. agglomerans cell suspension was added in a final concentration reached

0.5 mg dry wt/mL mixed thoroughly. The alginate-biomass mixture was then extruded through a

20 mL hypodermic syringe into 2% CaCl 2 solution and kept for 2 h at 40°C for bead formation. The resultant beads were 1.5 ± 0.2 mm diameter. The spherical beads were then rinsed thoroughly with deionized water and air dried and packed in the column.

In the same way, silica granules, charcoal, agar cubic and calcium alginate beads were prepared without adding cell biomass and used as the control.

Fig. 1 Laboratory plant for continuous bioremoval of lead.

Lead solution was pumped through the lower end of the glassware column at flow rate of 0.5 mL/min. The

2.7 Biomass Immobilization plant was cultivated at 40°C. The lead in the collection Four columns were packed with four different materials

vessel concentration was measured by atomic for the bacterial cell immobilization: white sand,

absorption spectrophotometer.

charcoal, agar and calcium alginate.

2.8 Desorption of Lead

2.7.1 White Sand Granules with 0.2-0.5 mm in diameter of pure silica

Three different desorption solutions were used; 0.1 were used after several time washing with DDW then

M Ethylene Diamine Tetra Acetic Acid-tetra sodium dried until constant weight was obtained. 60 g of these

salt, pH 7.5, 0.1 M Na 2 CO 3 , pH 11.7 and DDW. 50 mL granules were sterilized and packed in the column.

of desorption solution were pumped through the

2.7.2 Charcoal lead-loaded alginate bioreactor at flow rate of 0.5

50 g of 0.5-2.5 mm in diameter of charcoal granules mL/min. lead accumulated in the collection vessel was were washed several times with DDW, sterilized and

measured by atomic absorption spectrophotometer. packed in the column.

2.9 Factors Affecting Lead Bioremoval by Immobilized Thereafter, 20 mL of E. agglomerans (0.5 mg dry

Cells

wt/mL) was added to silica and charcoal-containing columns.

Different pH values (2, 3, 4, and 5), temperatures (20,

2.7.3 Agar

30, 40 and 50°C), time of contact (0.5, 1, 3, and 24 h)

0.44 g of agar was dissolved in 20 mL of normal were tested. The parameter that gives high bioremoval saline by boiling after a clear solution was obtained it

capacity was employed in the next experiment. was cooled to 45°C thereafter the E. agglomerans cells

2.10 Preferential Uptake of Lead by Immobilized Cells suspension was added in final concentration reached

A filter sterilized solution containing 100 mg/L solidify then it was cut into small cubic in order to be

0.5 mg dry wt/mL the whole suspension was left to

lead (as PbNO 3 ), 100 mg/L copper (as CuCl 2 ) and 100 packed in the column.

mg/L cadmium (as CdCl 2 ) was pumped through the

2.7.4 Calcium Alginate alginate bioreactor in order to investigate the Two grams of sodium alginate were dissolved in 50

influence of Co ions presence on lead uptake by mL of DDW with continuous and mixing until good

immobilized bacterial cells.

Bioremoval of Aquatic Environment Lead by Immobilized Cells of Enterobacter spp.

2.11 Effect of Physiological State of Bacterial Cells on the most efficient bioreactor in bioremoval of lead Lead Uptake

given that it was able to remove 98% (84.4 out of 86 (mg/L)) of lead ions. Consequently, alginate bioreactor

Killed E. agglomerans (10 min in boiling water bath) was adopted for subsequent experiments. Several

were used instead of live cells in the alginate bioreactor authors reported that alginate is inactive material that

to test the ability of dead microbial biomass to does not affect the cells nor considered as nutrient

uptake lead. besides it does not affected by metal toxicity in

addition to its low cost, therefore it considered the most Five Enterobacter species namely Enterobacter

3. Results and Discussion

appropriate immobilization material [11, 12]. agglomerans (E1, E2 and E3), E. gergoviae (E4) and E.

Present study did not found any significant sakazakii (E5) were isolated from the wastewater of the

differences (P < 0.05) between batch and continuous general state of batteries factory. Urrutia [8] reported

system in bioremoval of lead from its aqueous that gram negative bacteria are dominant in the

solutions (Fig. 3).

wastewater due to their well organized enzymatic

3.1 Lead Desorption

systems for the utilizing complex substances in addition to the presence of the outer membrane which

EDTA solution was the most efficient one in give them full protection against all external effectors.

recovery of lead from the four bioreactors since it was Table 1 shows the capacity of bacterial isolates in

able to recover 86%, 82.7%, 87.5% and 71.2% of bioremoval of lead from the aqueous solutions as E.

biosorbed lead on alginate, charcoal, white sand and agglomerans (E5) was the most efficient isolate since it

agar agar bioreactors, respectively, as it shown in Fig. 4. was able to remove 99.2% (76.7 ± 0.2 mg/L) from 77.3

This capacity could be attributed to the ability of mg/L lead initial concentration. However, there were

ETDA as a chelating agent to unbind the lead ions that insignificant differences (P > 0.05) among attached to the bacterial surface. Enterobacter isolates.

3.2 Factor Affecting Lead Bioremoval As they are negatively charged and abundantly

available, carboxyl, hydroxyl and phosphate groups

3.2.1 pH Effect

actively participate in the binding of metal cations. It can be seen clearly from Fig. 5 that lead bioremval Also, amine groups are very effective at removing

by E. agglomerans immobilized by alginate bioreactor metal ions, as it not only chelates cationic metal ions,

was highest at pH 4. Ricardo and Rdorigues [13] but also adsorbs anionic metal species via electrostatic

reported that the alginate beads contained 4.7 mmol/g interaction or hydrogen bonding [9]. Kang et al. [10]

COOH groups which suffered hydrolysis near pH 4. observed that amine groups attracted negatively

In general, increasing pH increases the overall charged chromate ions via electrostatic interaction.

negative charge on the surface of cells until all Results depicted in Fig. 2 revealed that alginate was

the relevant functional groups are deprotonated which

Table 1 Lead bioremoval capacity of bacterial species isolated from the wastewater of the general state of batteries factory.

Code Bacteria species Lead initial concentration (mg/L) Amount of biosorbed lead (mg/L) Bioremoval percentage (%)

E1 E. agglomerans 75.7 74.1 ± 0.047

E2 E. gergoviae 78.1 68.1 ± 0.124

E3 E. sakazakii 79.4 73.1 ± 0.22

E4 E. agglomerans 78.8 70.5 ± 0.235

E5 E. agglomerans 77.3 76.7 ± 0.249

Bioremoval of Aquatic Environment Lead by Immobilized Cells of Enterobacter spp.

Fig. 2 Efficiency of different bioreactors in bioremoval of lead.

Fig. 3 Lead bioremoval by immobilized and free cells of E. agglomerans.

100 87.5 86.8 DDW 90 82.7 78.3 EDTA

g 80 73 Na2CO3 ta

white sand

charcoal

agar agar

alginate

Fig. 4 Recovery of lead.

favors the electrochemical attraction and adsorption of metal speciation as well. Metal ions in solution undergo cations. Anions would be expected to interact more

hydrolysis as the pH increases. The extent of which strongly with cells with increasing concentration of

differs at different pH values and with each metal, but positive charges, due to the protonation of functional

the usual sequence of hydrolysis is the formation of groups at lower pH values. The solution chemistry

hydroxylated monomeric species, followed by the affects not only the bacterial surface chemistry, but the

formation of polymeric species, and then the formation

Bioremoval of Aquatic Environment Lead by Immobilized Cells of Enterobacter spp.

of crystalline oxide precipitates after aging [9,14].

3.2.3 Effect of Time of Contact

3.2.2 Effect of Temperature Biosorption process reached the equilibrium state There were no significant differences (P < 0.05)

within one hour given that E. agglomerans (E5) between different temperatures tested in lead biosorbed 93.8 mg/L. However, there were no bioremoval (Fig. 6). A matter confirmed that lead

significant differences (P < 0.05) after 24 hours of bioremoval is an exothermic reaction [15].

contact (Fig. 7).

ount g m (m

Fig. 5 Effect of pH on lead bioremoval by alginate bioreactor.

Tem perature °C

Fig. 6 Effect of temperature on lead bioremoval by alginate bioreactor.

Fig. 7 Effect of time of contact on lead bioremoval by alginate bioreactor.

Bioremoval of Aquatic Environment Lead by Immobilized Cells of Enterobacter spp.

3.3 Preferential Uptake of Lead by Immobilized Cells Environment International 30 (2004) 261-278. [6] J.G. Holt, N.R. Krieg, P.H. Sneath, J.T. Staley, S.T. Alginate bioreactor was able to bind ions other than

Williams, Bergey’s Manual of Determinative lead in the order Cu > Pb > Cd. Ability could be

Bacteriology, 9th ed., Williams and Wilikins Baltimore, ascribed to the high affinity of to bind various USA, 1994. [7] S. Ilhan, M.N. Nourbakhsh, S. Kilicarslan, H. Ozdag,

metal ions. Removal of chromium, lead and copper ions from In conclusion, E. agglomerans (E5), isolated from

industrial wastewater by Staphylococcus saprophyticus, the wastewater of general state of batteries industries in

Turkish Electronic Journal of Biotechnology 2 (2004) 50-57.

Baghdad-Iraq, showed an outstanding ability to [8] M. Urrutia, General bacteria sorption processes, in: J.

biosorpe lead ions either as a free cells or immobilized Wase, C. Forster (Eds.), Biosorpents for Metal Ions, by alginate. The process was affected by pH and time

Taylor and Francis Ltd, UK, 1997. course while the temperature has insignificant effect.

[9] K, Vijayaraghavan, Y. Yun, Bacterial biosorbents and biosorption, Biotechnology Advances 26 (2008) 266-291.

Most of lead ions were recovered using EDTA solution [10] S. Kang, J. Lee, K. Kim, Biosorption of Cr (III) and Cr (VI)

a matter emphasizes the reusable of the biosorbed ions. onto the cell surface of Pseudomonas aeruginosa, The present study confirmed that the designed lab plant

Biochemical Engineering Journal 36 (2007) 54-58 containing bacterial cells binding on calcium alginate [11] C. Bucke, Cell immobilization in calcium alginate, in: K. Mosbach (Ed.), Methods in Enzymology, Vol. 135, 1987,

succeeded in uptake all lead ions from all samples of

pp. 175-178.

industrial wastewater of batteries factory. [12] J.L. Gardea-Torresdey, J.L. Arenas, N.M. Francisco, K.J. Tiemann, R. Webb, Ability of immobilized cyanobacteria

References

to remove metal ions from solution and demonstration of [1] H. Shumate, G. Strandberg, J. Parrott, Biological removal

the presence of metalothionein genes in various strains, of metal ions from aqueous process streams,

Journal of Hazardous Substance Research 1 (1998) Biotechnology and Bioengineering Symposium 8 (1978)

1013-1016.

12-20. [13] R Ricardo, J. Rodrigues, Evaluation of dry protonated [2] F.P. Padilha, P. de França, A.C. da Costa, The use of waste

calcium alginate beads for biosorption applications and biomass of Sargassum sp. for the biosorption of copper

studies of lead uptake, Applied Biochemistry and from simulated semiconductor effluents, Bioresource

Biotechnology 143 (2007) 115-128. Technology 96 (2005) 1511-1517.

[14] H.L. Liu, B.Y. Chen, Y.W. Lan, Y.C. Cheng, Biosorption [3] H. Alluri, S. Ronda, V. Settalluri, J. Singh, Biosorption:

of Zn (II) and Cu (II) by the indigenous Thiobacillus An eco-friendly alternative for heavy metal removal,

thiooxidans , Chemical Engineering Journal 97 (2004) African Journal of Biotechnology 6 (2007) 2924-2931.

195-201.

[15] D.H. Cho, E.Y. Kim, Characterization of Pb [4] N. Ahalya, T.V. Ramachandra, R.D. Kanamadi, +2 biosorption Biosorption of heavy metals, Research Journal of

from aqueous solution by Rhodotorula glutinis, Chemistry and Environment 7 (2003) 71-78.

Bioprocess and Biosystems Engineering 25 (2003) [5] A. Malik, Metal bioremediation through growing cells,

271-277.

Journal of Life Sciences 5 (2011) 974-980

Differences in Browning Index and CIELAB Coordinates of the Two Grape Drying Processes, Traditional Sun-Drying and Chamber-Drying and during the Ageing of Pedro Ximenez Sweet Wine

María P. Serratosa, Ana Marquez, Azahara Lopez-Toledano, Manuel Medina and Julieta Merida Department of Agricultural Chemistry, Faculty of Sciences, University of Cordoba, Edificio Marie Curie, Campus de Rabanales,

Cordoba E-14014, Spain

Received: February 03, 2011 / Accepted: April 20, 2011 / Published: November 30, 2011.

Abstract: The color parameters during the Pedro Ximenez grape raisining, as well as the sweet wine aging process from the Montilla-Moriles grapes (Andalusia, Southern Spain), have been studied. Drying process of grapes was carried out by means of the traditional sun-drying method and an alternative chamber-drying method under 50ºC. Chamber-drying allows shorter drying time and

select grapes at a higher ripening degree. During raisining grape musts decreased in h ab (hue angle) and increased in C * ab (chroma). In comparative terms, the final values of hue were virtually identical in both types of drying, although differences were found in the final values of chromaticity, being lower in the chamber-drying method. Changes in the color parameters during aging were compared in commercial wines with different aging systems and without aging. Likewise, as a reference of traditional wine aging system, the color changes in wines with four aging degrees were also studied. Regarding to the commercial wines studied, it can be pointed out the wine aged without blends for 4 years significantly differed in the values of h

ab and C ab of the remaining wines, which show more similar values among them and in the data obtained for the wines aged by the traditional aging system.

Key words: Browning index, CIELAB, traditional sun-drying, chamber-drying method, aging degrees, sweet wine.