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Journal of Environment and Waste Management
Vol. 5(2), pp. 275-282, November, 2018. © ISSN: 0274-6999

Research Article
Optimization of Na-Alginate Immobilization Method for
Sulfide Oxidation Using Immobilized Thiobacillus Species
Ajit Kumar Surabhi1 and *Mohamed Ibrahim Elzagheid2
1,2Chemicaland Process Engineering Technology Department, Jubail Industrial College, Jubail Industrial city, 31961,
Kingdom of Saudi Arabia
The study was aimed to investigate the optimization of Na-alginate immobilization method for
biological sulfide oxidation process using an immobilized Thiobacillus species and the effects of
four factors including Na-alginate concentration, CaCl2 concentration, agitation speed and
amount of inoculum on sulfide conversion. The strength of sodium-alginate immobilization
method for Thiobacillus species was evaluated. For this purpose, experiments were designed by
a central composite design (CCD) and results were optimized by using response surface
methodology (RSM). Design of experiments (DOE) was used to model and optimize the
operational conditions. The central composite design (CCD) was very good for the optimization
of variables; the R2 value for the developed model was 0.91. The results and analysis showed the
optimized values for the sulfide oxidation. 95% Sulfide oxidation was achieved with optimized
values. Beside this a useful by- product was produced from waste effluents.
Keywords: Cell Immobilization, Sodium alginate, Sulfide, Sulfur-oxidizing bacteria, Thiobacillus.


Microbial Anaerobic reduction of sulfate or sulfite to sulfide al., 2012). In order to overcome these difficulties, biological
is known as a traditional process to remove sulfate for based sulfide removal methods can be a better choice.
several years (Tikhonova et al., 2006; Azabou et al., 2007). Due to their low temperature and low-pressure operational
As a result, much sulfide is discharged to natural water conditions, biological sulfide oxidation methods have more
bodies and much hydrogen sulfide gas (H2S) is leaked into advantages over conventional sulfide removal methods
the atmosphere. Certain industrial wastewaters also [Midha et al., 2012; Ramos et al., 2013; Zytoon et al., 2014;
contain sulfide that has severe toxic effects on ecosystems Toth et al., 2015). Several microorganisms have the ability
even at very low levels (Wiessner et al., 2005). to remove sulfur and sulfides from wastewater. Among
Wastewater effluents released by the various industries those are Acidithiobacillus and Thiobacillus species:
like crude oil refineries, petrochemicals, pulp and paper, Acidithiobacillus thiooxidans (Aroca et al., 2007),
tannery, and fertilizers also contains considerable Thiobacillus novellus (Cha et al., 1999), Thiobacillus
quantities of sulfur-contained emissions. These are thioparus, (Chung et al., 2000; Cox and Deshusses, 2002)
affecting environment severely, causing global warming and Thiobacillus denitrificans (Ma et al., 2006) and for
and land, air, and water bodies’ pollution. H2S is a toxic sulfide oxidation Thiobacillus denitrificans, Thiobacillus
and corrosive gas. It causes several environmental thioparus, Thiobacillus thiooxidans, and Thiobacillus
problems, and economical issues to processing industries ferrooxidans (Ramirez et al., 2009; Cardoso et al., 2006;
in the form of equipment corrosion (Vaiopoulou et al., Lee et al., 2006; Visser eta l., 1997).
2005; Li et al., 2009). Most of current commercial methods
of sulfide removal are physicochemical methods, namely
Amoco-cold based adsorption, Lurgi sulfreen, shell class *Corresponding Author: Mohamed Ibrahim Elzagheid,
off-gas treating process. Even though physicochemical Chemical and Process Engineering Technology
sulfide removal methods were successful, but their high- Department, Jubail Industrial College, Jubail Industrial city,
energy, chemical, and catalyst consumptions, high 31961, Kingdom of Saudi Arabia. Email:
operating costs, and huge capital investments made them,, Tel:
expensive to operate (Ahmad et al., 2017; Liang et al., 00966133425428. Co-Author Email:,
2015; Eow, 2002; Pandey and Malhotra, 1999; Midha et Tel: 00966133425424

Optimization of Na-Alginate Immobilization Method for Sulfide Oxidation Using Immobilized Thiobacillus Species
Surabhi and Elzagheid 276

Sulfide oxidation reaction pathway in Thiobacillus species
follows the main oxidation pathway of S2---->S0---->SO32--
--->SO42-. It is often not easy to control and separate these
reactions that carried out by microorganism (Midha et al.,
2012; Suzuki, 1974 and 1999). Due to this, it is essential
to stop formation of sulfates, by using an immobilization
method which may help avoiding formation of sulfates,
safeguards the cells from wash away, and prevents them
from contacting harmful compounds (Li et al., 2009). The
use of immobilized cells is increasing in food production,
medicine, biofuels, chemical industries, industrial
wastewater treatment, and in textiles (Midha et al., 2012;
Toth et al., 2015; Siripattanakul et al. 2008; Mirzaei et al., Figure 1: Thiobacillus species immobilized in Na-
2014). Several natural and synthetic support materials are alginate beads
available for immobilization such as agar, alginate,
agarose, kappa-carrageenan, and synthetic like polyvinyl The beads were prepared with a peristaltic pump using a
alcohol (Mirzaei et al., 2014). Immobilization techniques pipe of 2 mm diameter in CaCl2 solution. The 3 % CaCl2
like carrier bonding, adsorption, cross-linking, and cross-linking solution was prepared by dissolving 6 gm of
encapsulation are being used widely in industries and in CaCl2 in 150 ml of double distilled water. The beads were
researches (Li et al., 2009; Toth et al., 2015; Mirzaei et al., allowed in cross-linking solution for 3 hours. Later beads
2014; Krishnakumar et al., 2005).There were several were transferred into 150 ml of maintenance medium
approaches to immobilized cell biological sulfide oxidation, containing NH4Cl, 4 g/l; MgSO4.7H2O, 1 g/l; KH2PO4, 2 g/l;
but such techniques have failed to explain the effects of and 10 ml of trace element solution for overnight. The whole
operational variable interactions, and difficult to provide process was carried out in aseptic conditions to prevent any
optimum operation variables (Habeeb et al., 2016). The possibility of contamination.
aim of this study is utilize central composite design (CCD)
and design of experiments (DoE) under response surface Experimental set-up: Experiments were conducted in
methodology (RSM). Response surface methodology 500 ml laboratory shake-flask reactor. Prior to the
(RSM) is a statistical approach and is a mathematical experiment, the conical flasks were stocked with media,
technique that can explain the relation and interactive and sterilized with other accessories at 121 oC for 15
effects between several independent variables and one or minutes. After cooling the media, the shake-flask reactor
more responses. RSM is a very effective tool for was inoculated with the Thiobacillus organism culture. The
optimization (Kalantari et al., 2017). The current study operation was carried out in batch wise, the temperature
focuses on elevation and optimization of process was maintained in a shaking oven at 25 oC, halogenation
conditions of sodium alginate immobilization method for was achieved by slow shaking and the aeration was
sulfide removal from waste effluent water stream provided with an air pump.
biologically using immobilized Thiobacillus species.
Analytical methods: For sample analysis standard
methods, APHA Standard methods for the examination of
MATERIALS AND METHODS water and waste water 20th edition was used for the
analysis of pH, temperature, sulfide, sulfate in the liquid
Collection, isolation, and immobilization of the microbial media.
species were carried out as described by Pavichandra et
al (2007, 2008, and 2009) and Himabindu et al (2006). Experimental design and optimization: The optimum
Accordingly, sulfide oxidizing bacteria was isolated from levels of parameters of Na-alginate immobilization method
aerobic sludge, which was collected from wastewater for the maximum oxidation of sulfide by Thiobacillus
treatment plant. The sludge is then kept for activation by species determined by means of RSM. The RSM consists
mixing sulfide-oxidizing bacteria (SOB), Thiobacillus of a group of empirical techniques devoted to the
enrichment media. After adapting the sludge to evaluation of relationships existing between a cluster of
Thiobacillus media for a period of one month the sludge controlled experimental factors and measured responses
was used as a source for the isolation (SOB) of according to one or more selected criteria. According to
Thiobacillus species. The Thiobacillus species cells were this design, the total number of treatment combinations
immobilized in Na-Alginate by entrapment method, Figure was 2^k + 2*k + n0 where k is the number of independent
1. Bacterial cell pellet was weighed and 20 mg of cells variables and n0 is the number of repetitions of the
were added to sodium alginate solution. The 3% sodium experiments at the center point. Based on the best results
alginate solution was prepared by adding 3 gm of high of one at a time approach, four critical parameters of c-
viscous sodium alginate in 100 ml of Thiobacillus species alginate immobilization method medium were selected and
growth media by continuous stirring till the sodium alginate evaluated for their interactive behaviors by using a
was completely mixed in solution.
Optimization of Na-Alginate Immobilization Method for Sulfide Oxidation Using Immobilized Thiobacillus Species
J. Environ. Waste Manag. 277

statistical approach. The levels of four variables viz. Na- Table 2: Design of experiments by full factorial Central
alginate concentration, 3% (x1); NaCl2 3% (x2); inoculum Composite Design (CCD) for RSM Studies
size, 6% (x3); and agitation 150 rpm (x4) were selected Run Run
x1 x2 x3 x4 x1 x2 x3 x4
and each of the variables was coded at five levels –2, –1, No. No.
0, 1, and 2 by using Equation (1). 1 -1 -1 -1 -1 16 1 1 1 1
2 1 -1 -1 -1 17 -2 0 0 0
xi = Xi – X0/ΔX-----(1) 3 -1 1 -1 -1 18 2 0 0 0
4 1 1 -1 -1 19 0 -2 0 0
For statistical calculations, the variables Xi were coded as 5 -1 -1 1 -1 20 0 2 0 0
xi according to the following transformation (Himabindu et 6 1 -1 1 -1 21 0 0 -2 0
al., 2006). The range and levels of the variables in coded 7 -1 1 1 -1 22 0 0 2 0
units for RSM studies are given in Table 1. 8 1 1 1 -1 23 0 0 0 -2
9 -1 -1 -1 1 24 0 0 0 2
Table 1: Range and levels of the variables used in RSM
10 1 -1 -1 1 25 0 0 0 0
and optimization studies
11 -1 1 -1 1 26 0 0 0 0
Variables -2 -1 0 +1 +2 ∆x
12 1 1 -1 1 27 0 0 0 0
Na-alginate 1 2 3 4 5 1
13 -1 -1 1 1 28 0 0 0 0
concentration, %, x1, w/v
14 -1 1 1 29 0 0 0 0
CaCl2,%,x2, v/v 1 2 3 4 5 1
15 -1 1 1 1 30 0 0 0 0
Inoculum size,%, x3, v/v 2 4 6 8 10 2
Agitation, rpm, x4 50 100 150 200 250 50
The results of CCD experiments for studying the effect of
Where xi is the dimensionless coded value of the variable three independent variables are presented along with the
Xi, X0 the value of the Xi at the center point, and ΔX the mean predicted and observed responses in Table 3. The
step change. The following quadratic model 2 explained regression equations obtained after the ANOVA gave the
the behavior of the system. level of Thiobacillus species growth as a function of the
initial values of Na2S2O3.5H2O, 6 gm, CaCO3, 3 gm, NH4Cl,
Y  0    i * x i   ii * x i   ij * x ij ---- (2)
2 0.4 gm, CaCl2, 0.5 gm, KH2PO4, 2 gm and (NH4)2SO4 3
gm. The application of RSM yielded the following
regression equation, which is empirical relationship
Where Y, is the predicted response, β0 the intercept term, between Thiobacillus species growth (Y) and the test
βi the linear effect, βii the squared effect, and βij is the variables in coded unit.
interaction effect. The full quadratic equation for four
factors is given by model 3. Table 3: Results of Design of Experiments by Central
Composite Design (CCD) for RSM Studies
Y = β0 + β1 x1 + β2 x2 + β4 x3 + β4 x4 + β11 x1*x1 +β12 Sulfide Sulfide Sulfide Sulfide
x1*x2 + β13 x1*x3 + β14 x1*x 4 + β22 x2*x2 + β23 x2*x3 + β24 Run oxidation oxidation Run oxidation oxidation
x2*x 4 + β33 x3*x3+ β34 x3*x4 + β44 x4*x4 ----- (3) No. % % No. % %
measured Predicted measured Predicted
Several experimental designs have been considered for 1 92 92.54167 16 73 91.3333
studying such models and central composite design (CCD) 2 86 85.875 17 86 72.79167
was selected. For this study, a full factorial central 3 97 80.375 18 72 82.95833
composite design with eight star points and six replicates 4 86 79.83333 19 80 69.29167
at the central points were employed to fit the second order 5 85 69.20833 20 72 80.45833
polynomial model, and 32 experiments were required for 6 80 74.66667 21 86 85.95833
this procedure. STATISTICA by Stat Soft Inc. software 7 82 74.16667 22 88 85.79167
was used for regression and graphical analysis of the data 8 77 76.875 23 92 80.79167
obtained. In order to search for the optimum combination 9 80 73.375 24 86 94.95833
of major critical parameters of the sodium alginate 10 72 80.83333 25 80 77.33333
immobilization method for the sulfide oxidation,
11 68 82.33333 26 77 77.33333
experiments were performed according to the CCD
12 70 87.04167 27 76 77.33333
experimental plan shown in Table 2.
13 91 76.16667 28 74 77.33333
14 88 86.875 29 79 77.33333
15 70 93.375 30 78 77.33333

Optimization of Na-Alginate Immobilization Method for Sulfide Oxidation Using Immobilized Thiobacillus Species
Surabhi and Elzagheid 278

RESULTS AND DISCUSSIONS of each coefficient was determined by Student’s t-test and
p-values, which are listed in Table 4 and 5. The larger the
Optimization of Na-alginate immobilization method for magnitude of the t-value and smaller the p-value, the more
sulfide oxidation: significant is the corresponding coefficient. This implies
that there are significant linear effects of medium variables
The results of composite design experiments (CCD) matrix on sulfide oxidation x1 (p<0.00134), x2 (p<0.00061) and
for percentage (%) of observed and measured biological x4 (p<0.000064), the interactive effects of x2x3 (p <
sulfide conversion by sulfide oxidation bacteria (SOB) 0.020061) x2x4 (p < 0.020061) x3x4 (p < 0.020061) and
thiobacillus species, and the evaluated model was quadratic effects of x3 (p < 0.003057) and x4 (p <0.00057).
represented by the below given Equation 4. By applying The model F-value of 10.83699, and values of prob > F
multiple regression analysis on the experimental data, the (<0.05) indicated that the model terms are significant.
experimental results of the CCD design were fitted with a
second order full polynomial equation. The empirical Table 4: Model Summary and Analysis of Variance
relationship between sulfide oxidation (Y) and the four test (ANOVA) for the Quadratic Model
variables in coded units obtained by the application of Degree
RSM is given by Equation 4. Source of Sum of of Mean Prob-
Variations Squares freedom Square F-Value ability
Y = 77.33 -2.54* x1 -2.79* x2 -0.04* x3 -3.54* x4 +0.13* Regression 1526.45 14 109.0321 10.83699 1.99E-05
x1x1 + 0.68* x1x2 + 0.81* x1x3 + 1.31* x1x4 -0.61* x2x2 - Residual 150.9167 15 10.06111
2.06* x2x3 -3.06* x2x4 + 2.13* x3x3 + Total 1677.367
4.31* x3x4 + 2.63* x4x4----- (4) R=0.953954, R2 = 0.910028, Adjusted R2 = 0.826053,
CV=13.63 %
Where Y is sulfide oxidation in %, is response and x1, x2,
x3, and x4 are the coded values of the test variables, Na- Table 5: Model Coefficients Estimated by Multiple
alginate concentration, 3% (x1); CaCl2, 3% (x2); inoculum Linear Regressions (Significance of Regression
size, 6% (x3); and agitation, 150 rpm (x4). The coefficient Coefficients)
of determination value (R2) was calculated as 0.910028 for
sulfide oxidation (model summary, Table 4 and 5), Standard-
indicating that the statistical model can explain 91 % of ized Compu-
variability in the response. The R2 value is always between Unstandardized Coeffi- ted
0 and 1. The closer the R2 is to 1.0, the stronger is the Model Coefficients cients t-values p-values
model and the better it predicts the response, and higher B Std. Error Beta
degree of co-relation between observed and predicted (Cons-
values. In this case, the value of the determination tant) 77.33333 1.294933 59.71994 2.97E-19
coefficient (R2 = 0.910028) indicates that the model does x1 -2.54167 0.647467 -0.30403 -3.92556 0.001349*
not explain only 9 % of the total variations. The adjusted x2 -2.79167 0.647467 -0.33393 -4.31168 0.000617*
R2 value corrects the R2 value for the sample size and for x3 -0.04167 0.647467 -0.00498 -0.06435 0.949539
the number of terms in the model. The value of the x4 -3.54167 0.647467 -0.42364 -5.47004 6.46E-05*
adjusted determination coefficient (Adj R2 = 0.826053) is x1x1 0.135417 0.60565 0.017744 0.223589 0.826094
also high to advocate for a high significance of the model. x1x2 0.6875 0.792981 0.067146 0.866981 0.399609
If there are many terms in the model and the sample size x1x3 0.8125 0.792981 0.079354 1.024614 0.321786
is not very large, the adjusted R2 may be noticeably x1x4 1.3125 0.792981 0.128187 1.655146 0.118661
smaller than the R2. Here in this case the adjusted R2 value x2x2 -0.61458 0.60565 -0.08053 -1.01475 0.326314
is 0.826053, which is lesser than the R2 value of 0.910028, x2x3 -2.0625 0.792981 -0.20144 -2.60094 0.020061
these values are in good agreement with R2 =0.875 value x2x4 -3.0625 0.792981 -0.2991 -3.86201 0.001536
reported by Olmez (2009). At the same time, a relatively x3x3 2.135417 0.60565 0.279811 3.525829 0.003057
lower value of the coefficient of variation (CV = 13.63%) x3x4 4.3125 0.792981 0.421187 5.438337 6.86E-05
indicates a better precision and reliability of the carried out x4x4 2.635417 0.60565 0.345328 4.351389 0.00057
The results of multiple linear regressions conducted for the
Response surface methodology (RSM) approach was second order response model are given in Table 5. The
applied for identifying the significant factor. The ANOVA significance of each co-efficient that was determined by t
has conducted for the second order response surface & p-values, also listed in Table 5. The larger the value of
model and the results are given Table 4 and 5. The the t-value & smaller the p-value are more significant. The
Analysis of Variance (ANOVA) is statistical method used RSM method is an effective tool and stepwise procedure
to compare the means of two or more groups, The ANOVA in finding the optimum parameters. The regression model
provides complete information on model accuracy and developed can be represented in the form of contour plots.
significance, and identifies the most significant factors that The 2-D response surface and sulfide oxidation contour
influence the process (Zhou et al., 2010). The significance plots demonstrate the effects of factors on sulfide
Optimization of Na-Alginate Immobilization Method for Sulfide Oxidation Using Immobilized Thiobacillus Species
J. Environ. Waste Manag. 279

conversion efficiency are shown in Figures 2-7 at four the response variable. Figures 2, 3 and 4 show the trend
combinations. Each contour curve represents an infinite of sulfide oxidation in % with the variation in sodium
number of combinations of two test variables with the other alginate with the variation in sodium chloride
two maintained at their respective 0 level. The counter concentration, inoculum size and agitation speed in the
plots show the relationship between the sodium alginate range of -2 to +2 in coded units while the remaining other
with the variation in sodium chloride concentration, two respective variables were at their respective zero
inoculum size and agitation speed variables settings used levels. Figures 5 and 6 show the effect of calcium chloride
for an optimum sulfide conversion. The darker red regions concentration on the sulfide oxidation, as the
of the counter plots indicate that higher sulfide concentration of CaCl2 increased the sulfide oxidation was
conversions, while the other color regions indicate that increased. These plots demonstrate that the sulfide
lower sulfide conversions. oxidation is dependent on linear effects of Na- Alginate (Li
et al., 2009; Kalantari et al., 2017). A similar trend was also
The contour plots described by the regression model were observed for CaCl2 and agitation combination which
drawn to illustrate the effects of the independent variables, shown in Figure 7.
and combined effects of each independent variable upon

Figure 2: Contour plot of sulfide oxidation (%): Figure 3: Contour plot of sulfide oxidation (%):
the effect of Na-alginate and CaCl2 on sulfide oxidation. the effect of Na-alginate and Inoculum size on sulfide
Other variables are held at zero level oxidation. Other variables are held at zero level

Figure 4: Contour plot of sulfide oxidation (%): the Figure 5: Contour plot of sulfide oxidation (%): the
effect of Na-alginate and agitation on sulfide oxidation. effect of CaCl2 and Inoculum size on sulfide oxidation. Other
Other variables are held at zero level variables are held at zero level

Optimization of Na-Alginate Immobilization Method for Sulfide Oxidation Using Immobilized Thiobacillus Species
Surabhi and Elzagheid 280

Figure 6: Contour plot of sulfide oxidation (%): the Figure 7: Contour plot of sulfide oxidation (%):
effect of CaCl2 and agitation on sulfide oxidation. the effect of Inoculum size and agitation on sulfide
Other variables are held at zero level oxidation. Other variables are held at zero level

CONCLUSIONS Thiobacillus thioparus and Acidithiobacillus
thiooxidans. Electronic Journal of Biotechnology, 10
This study shows that immobilized Thiobacillus species as (4): 514–520. doi: 10.2225/vol10-issue4-fulltext-6.
sulfur oxidizing bacteria (SOB) can oxidize the sulfide into Azabou, S., Mechichi, T., Patel, B. K. C., Sayadi, S. (2007).
elemental sulfur. Optimization of sulfide oxidation Isolation and characterization of amesophilic heavy-
conditions was studied by using response surface method metals-tolerant sulfate-reducing bacterium
(RSM) of optimization by central composite. Design of desulfomicrobium sp. froman enrichment culture using
experiments (DOE) was used to model and optimize the phosphogypsum as a sulfate source, Journal of
operational conditions. The central composite design Hazardous Materials, 140: 264–270. doi:
(CCD) was very good for the optimization of variables; the 10.1016/j.jhazmat.2006.07.073.
R2 value for the developed model was 0.91. The results Cardoso R. B., Sierra-Alvarez R., Rowlette P., Flores E.
and analysis showed the optimized values for the sulfide R., Gomez J., Field J. A. (2006). Sulfide oxidation under
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Cha, J. M., Cha, W. S. and Lee, J.-H. (1999). Removal of
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ACKNOWLEDGMENT Thiobacillusnovellus SRM, sulphur-oxidizing bacteria,
Process Biochemistry, 34 (6-7): 659-665.
Authors of this article thank Jubail Industrial College doi:10.1016/S0032-9592 (98)00139-3.
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Accepted 13 November 2018

Citation: Surabhi AK, Elzagheid MI (2018). Optimization
of Na-Alginate Immobilization Method for Sulfide Oxidation
Using Immobilized Thiobacillus Species. Journal of
Environment and Waste Management 5(2): 275-282.

Copyright: © 2018 Surabhi and Elzagheid. This is an
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Optimization of Na-Alginate Immobilization Method for Sulfide Oxidation Using Immobilized Thiobacillus Species