Cd(II) biosorption using bacterial isolates from sawdust: optimization via orthogonal array Taguchi method

COPYRIGHT © The Author(s) Published under a Creative Commons Attribution 4.0 International Licence (CC BY 4.0) Orthogonal array of Taguchi experimental design with L16 four-level factors: pH (2–8), temperature (303–333 K), time (1–4 h), inoculum concentration (5–20 v/v) and Cd(II) initial concentration (50–200 mg/L) was applied to optimize Cd(II) biosorption from aqueous solution via bacterial isolates from sawdust. The optimum conditions were found to be 4, 303 K, 4 h, 15 v/v % and 50 mg/L for pH, temperature, time, inoculum concentration and Cd(II) initial concentration, respectively. A confirmatory experimental run at these conditions revealed 99.53% Cd(II) removal. Fourier transform infrared revealed the presence of –OH on the bacterial surface enhancing Cd(II) biosorption. The presence of small cavities on the bacterial surface with a porous inner multilayer was shown by scanning electron microscopy analysis. Proposed biosorption mechanisms were electrostatic interaction, surface complexation and ion exchange. In conclusion, bacterial isolates from sawdust could effectively be applied as biosorbent for Cd(II) removal from aqueous solution. Cd(II) biosorption using bacterial isolates from sawdust: optimization via orthogonal array Taguchi method


INTRODUCTION
Water is among the natural resources needed for germination of crops, to achieve sustainable food production via irrigation (Hristov, 2014). Various forms of anthropogenic activities contaminate this natural resource when humans engage in industrial activities to manufacture end-products for consumption (Abdulla et al., 2019). This adversely affects the water and makes it unsuitable for agricultural purposes (fishing and irrigation). Sewage sludge and industrial effluents discharged into water bodies contain contaminants. Among these are heavy metals such as cadmium, lead, chromium, mercury and arsenic, whose presence at low concentrations stunts plant growth by altering its biochemical and physiological development, leading to chlorosis, root growth inhibition and plasma membrane damage (Bouazizi et al., 2010;Mkadmi et al., 2018;Nagendrappa et al., 2010). Declining plant growth decreases yield, which affects food supplies. Remediation of heavy metal contaminated water is thus important for food security.
Previously, two-level Plackett-Burman factorial design was used as an optimization tool for removal of Pb 2+ using Gelidium amansii (El-Naggar et al., 2018a), and Cr 5+ and Cu 2+ using Aspergillus terreus (Abou-Taleb et al., 2017), while response surface methodology has been applied for removal of phenol using Pseudomonas putida (Sridevi et al., 2011) and of Pb 2+ using Aspergillus niger (Amini et al., 2008) from aqueous solutions. However, application of Taguchi experimental design in different areas of wastewater treatment have gained serious attention over other experimental design methods due to its improved quality, and robust design (Daneshvar et al., 2007;Mousav et al., 2007;Barrado et al., 1996;Engin et al., 2008). Also, studies have shown the high resistance of Pseudomonas aeruginosa to pollutants including heavy metals, antibiotics, organic solvents and detergents (Haritash et al., 2009;Chellaiah, 2018).
This study applied L 16 four-level factors (4^5) orthogonal array of Taguchi experimental design for investigating optimization of Cd(II) biosorption from polluted water using bacteria isolates from waste sawdust. The analysis of mean (ANOM) approach was adopted for statistical optimization of process parameters (pH, temperature, time, inoculum concentration and Cd(II) salt initial concentration) that influence Cd(II) biosorption from aqueous solution. Analysis of variance (ANOVA) technique was applied to check the percentage contribution of individual process parameters to the biosorption process. An experiment was conducted to correlate the predicted optimized condition by the Taguchi experimental design. Few investigations have been conducted in the past on application of bacteria isolates from sawdust as active bio-agents for the removal of Cd 2+ from aqueous solution. Characterization was done using FTIR and SEM.

Waste sawdust collection
A sawdust sample was collected from New Garage Saw Milling Industry, Ibadan, Oyo State, Nigeria. Dirt was removed from the sawdust by hand picking. It was then passed through a mesh size of 1.18 mm to obtain a smaller particle size. The sawdust was then washed thoroughly in distilled water and sieved. About 20 kg of sieved particle was later soaked in a bucket containing water at room temperature for 14 days to activate bacterial growth.

Isolation and characterization of bacteria
The dilution plate method was adopted to isolate and characterize bacterial isolates from the fermented sawdust. Ten grams of fermented sawdust was added to 90 mL distilled water. Twentythree grams of nutrient agar (Lifesave biotech, USA) (peptic digest of animal tissue = 5.0 g/L, beef extract = 1.50 g/L, yeast extract = 1.50 g/L, sodium chloride = 5.0 g/L, agar = 15.0 g/L and final pH at 25°C = 7.4 ± 0.2) were dissolved in 1 000 mL of distilled water and continuously stirred until homogeneity was ascertained. Ten different samples containing 9 mL of diluted sawdust and 10 mL of nutrient agar each were set up under aerobic conditions in a petri dish and placed in an incubator at 37°C for 5 days. The discrete bacterial colonies were sub-cultured and sterilized. Stock cultures were then prepared from the pure cultures and stored at 4°C for further laboratory work.
Bacterial isolates were characterized using the sugar fermentation test. The method described by Cheesbrough (1985) was adopted to conduct a motility test. The methods described by MacFadden (2000) were used to perform biochemical tests. The various tests executed include motility, methyl-red, catalase, glucose, sucrose, mannose, lactose, pigment, oxidase, gram reaction and citrate.

Preparation of Cd(II) solution
One gram of 1 M Cd(NO 3 ) 2 •3H 2 O salt was dissolved in 1 L of distilled water to form simulated stock solutions in a 1 000 mL round bottom flask. Different initial concentrations of Cd(II) solution were prepared in 200 mL conical flasks for batch biosorption process using a Taguchi experimental design, as shown in Table 1.

Batch biosorption process
A temperature-controlled stirrer (Stuart heat-stirrer SB162) was used to investigate the batch biosorption of Cd(II) from solution using 5-day cultured bacterial isolates. The values for pH, temperature, time, inoculum concentration and Cd(II) salt initial concentration investigated for the batch process were based on the values specified by L 16 four-level factors (4^5) orthogonal array of Taguchi experimental design (Ghani et al., 2013) as presented in Table 2 (16 experimental runs). The pH of the solution was varied using 1 M HCl and 1 M KOH aqueous solutions. Constant values of 100 mL of Cd(II) salt in 250-mL flasks and 130 r/min were used throughout the experiment. Whatman PTFE filter paper was used to separate the filtrate from the residue. Cd(II) concentration was measured using an atomic absorption spectrometer (AAS Buck Scientific 210 VGP, USA). The concentrations of Cd(II) in the solution after a specified time were measured at a dilution factor of 50 and average values were recorded. The potential of bacterial isolates from sawdust to remove Cd(II) from solution was determined by measuring the percentage of Cd(II) adsorbed using Eq. 1.
where C o is the initial concentration of Cd(II) (mg/L) and C t is the final concentration of Cd(II) (mg/L) after each experimental run.

Optimization studies using analysis of mean technique
In order to execute optimization studies for Cd(II) biosorption from aqueous solution using bacterial isolates from sawdust, 5 operation factors were examined such that each of them was set at 4 different levels, as presented in Table 1. The experimental conditions for optimum biosorption of Cd(II) were determined using L 16 orthogonal array of Taguchi experimental design as presented in Table 2. Signal-to-noise (S/N) ratio was calculated using Eq. 2 (Pundir et al., 2018) for statistical analysis to maximize the process conditions for optimum biosorption. = S/N ratio value with factor F at level i in its j th appearance (j =1, 2,3 and 4) and n Fi = number of appearances of factor F in level i.

Determination of factor percentage contribution using analysis of variance
Analysis of variance (ANOVA) was employed to determine the percentage contribution of each process factor (P F ) to Cd(II) biosorption using Eq. 4: where SS F , DOF F , V Er and SS T are factorial sum of squares, degrees of freedom of each factor, variance of error and total sum of squares expressed as Equations 5, 6, 7 and 8, respectively.
where m = number of experiments executed n = number of replications of each experiment L = number of levels of each factor = cumulative average of Cd(II) percentage removal with a certain factor F at k th level (expressed as Eq. 9) = cumulative average of Cd(II) removed from solution (expressed as Eq. 10).
where n Fk = number of factor F appearances at level k factor F at level k in its j th appearance sequence ( j = 1, 2, 3….n Fk ).
where R i = percentage Cd(II) removed from solution in replication experiment i executed under the same experimental conditions for each test run.

Characterization of the bacterial cell surface
The active functional groups present on the surface of the bacterial isolates were investigated using Fourier-transform infrared spectroscopy (Nicolet iS10 FT-IR Spectrometer, USA) within a wavelength of 400-4 000 cm -1 . Beams of infrared were directed at the sample. The quantity and frequencies at which samples absorbed IR light were measured. The sample molecular identities were determined using the reference database.
The morphology of the bacteria was studied using a scanning electron microscope (SEM-JEOL-JSM 7600F) operated under high-vacuum evaporation at 5 000×, 15 kV. The sample was coated using a low-vacuum sputter platinum coating and then placed in a relative high-pressure chamber. The electron optical column was differentially pumped to ensure that the vacuum was kept adequately low. Secondary electron signal amplification was provided by the high-pressure region around the sample which neutralizes the charge. Due to the field emission gun's (FEG) ability to produce high primary electron brightness, low-voltage SEM was used in the FEG-SEM.

RESULTS AND DISCUSSION
Calculation of average percentage Cd(II) removal and signal-to-noise ratio at factor levels Equation 1 was used to determine the percentage of Cd(II) removed from aqueous solution (A i ) for each of the experimental tests, which were replicated for i = 1, 2, 3 and 4 for specified values of pH (2, 4, 6 and 8), temperature (303, 313, 323 and 333 K), time (1, 2, 3 and 4 hrs), inoculum concentration (5,

Solution pH
The effect of solution pH on percentage of Cd(II) removed from aqueous solution and signal-to-noise ratio was investigated (Fig. 1). It was observed that the percentage of Cd(II) removed from solution decreased from 89.29 to 86.87% when the solution pH was increased from 2 to 6. This could be attributed to inactivity and death of some cells (reduction in population) of the bacterial isolates due to the presence of strong acid (Yan et al., 2003). Also, functional groups on the outer surface of the bacteria become positively charged at lower pH, thereby creating repulsion between their surface and Cd(II) in solution which reduces heavy metal removal from solution (Yan et al. 2003

Reaction temperature
The effect of temperature on the average percentage Cd(II) removed from solution (in the presence of bacterial isolates) and signal-to-noise ratio is shown in Fig. 2. The result revealed a decrease in the average percentage of Cd(II) removed from solution from 91.39 to 85.88% when the reaction temperature was increased from 303 to 333 K. This resulted from damage to proteins present in bacterial colonies, which in turn reduced their growth rate and metabolic activities at this higher temperature (Huang et al. 2016). Research has revealed that bacterial efficacy in removing heavy metals from solution varies from one to another under different temperature conditions Javanbakht et al., 2014). Some studies have produced similar results to the current study (Yan et al., 2003;Sankarammal et al., 2014). Figure 3 presents the variation of average Cd(II) uptake from solution by bacterial isolates and signal-to-noise ratio at different levels of reaction time. As the reaction time increases, more of the Cd(II) was removed from solution by the bacteria as a result of an increase in their growth resulting from the formation of more colonies. However, partial equilibrium was attained after 3 h, substantiating the efficiency of the bacterial colonies in removing Cd(II) from aqueous solution as the percentage of heavy metal removed was almost constant. A high importance of reaction time in biosorption processes for heavy metals has been reported in several studies (Popoola, 2019a;Rao et al., 2006). Various equilibrium times attained for Cd(II) removal from solution have been reported, as 45 min (Haq et al., 2015), 90 min (Popoola, 2019b) and 120 min (Singh et al., 2000).

Inoculum concentration
A plot of inoculum concentration against average Cd(II) removed and S/N ratio is shown in Fig. 4. The average percentage Cd(II) removed from aqueous solution increased with an increase in the concentration of inoculums. A lower percentage removal of Cd(II) (87.97%) recorded at lower inoculum concentration (5 v/v %) resulted from lower populations of bacteria available to remove the heavy metal from solution. However, greater bacterial growth was observed at increased inoculum concentration (20 v/v %), which facilitated the biosorption of Cd(II) from solution.
The experimental values obtained for the average percentage of Cd(II) removed from water using bacterial isolates from sawdust at different factor levels are presented in Figs 1-5. A general predictive model (developed using the Taguchi approach) which could be used to establish an empirical relationship between the average percentage of Cd 2+ removed from water and independent process variables is stated as Eq. 11: where A, B, C, D and E represent pH, temperature, time, inoculum concentration and Cd(II) initial concentration, respectively. The values indicated within the square brackets represent levels of the corresponding model terms.

Application of analysis of variance to evaluate factor percentage contribution
The value of R T was calculated using Eq. 10 to analyse the percentage contribution of pH, temperature, reaction time, inoculum volume and Cd(II) initial concentration towards Cd(II) removal from aqueous solution by bacterial isolates. The calculated value of R T was 88.60. The factorial sum of squares, SS F , for each factor (Table 6), was calculated via substitution of R T and R k F into Eq. 5. The value of total sum of squares (SS T ) calculated via Eq. 8 was 535.85. The value of variance of error (V Er ) calculated by substituting SS F and SS T into Eq. 7 was 4.34. Thus, substitution of DOF F = 3, SS T and SS F into Eq. 4 gives the percentage contribution of each factor (ρ F ) ( Table 6) to Cd(II) removal from aqueous solution using bacterial isolates from sawdust.
The result obtained (Table 6) revealed the influence of each of the examined parameters (measured as a percentage) on Cd(II) biosorption from aqueous solution by bacterial isolates from sawdust. The order of influence was observed to be Cd(II) initial concentration (33.87%) > time (26.14%) > inoculum concentration (20.05%) > temperature (8.64%) > pH (6.07%). Studies have shown that the higher the initial concentration of heavy metal, the lower its removal from solution by adsorbents (Popoola, 2019b;Amini et al., 2008). Thus, initial concentration of a heavy metal greatly influences the rate of its biosorption from solution. This is because a high concentration of Cd(II) in solution hinders the growth of bacteria which adversely affects its efficiency in removing Cd(II) from solution. Hence, this affirms that the parameter with the greatest influence is Cd(II) initial concentration.
A recent study did not consider reaction time as part of the factors that affect removal of nickel and copper using Aspergillus sp. (Pundir et al., 2018). In the current study, it was revealed that the longer the inoculum stays in solution, the more the Cd(II) was removed (Fig. 3). Thus, the influence of reaction time cannot be underestimated; it ranks second, with a percentage contribution of 26.14%.
Also, the influence of inoculum concentration cannot be overlooked as more bacterial colonies are formed with increases in the initial concentration of inoculum. The rate of Cd(II) removal from solution was greatly affected by increasing bacterial isolate populations, leading to an increase in percentage removal for the heavy metal. Thus, inoculum concentration was revealed to be the third most influential parameter, with a percentage contribution of 20.05%. Similar studies have revealed similar results Hamza et al., 2010).
Nevertheless, temperature is another factor that also influences the removal of heavy metal from solution using microorganisms (Ding et al., 2012). In this study, the percentage contribution of temperature to the biosorption process was revealed to be 8.64% and it occupied 4 th position. Microbial growth is hindered when subjected to higher temperatures as the protein nature of bacteria is altered under these conditions (Sankarammal et al., 2014). Thus, the higher the temperature, the lower the bacterial efficiency and the more adversely removal of Cd(II) from solution is affected.
Influence of solution pH on heavy metal removal from solution using microorganisms has been documented previously (Dursun et al., 2003;Aksu et al., 2000). The present study revealed pH as having the lowest contribution (5 th position), with a percentage influence of 6.07% on Cd(II) biosorption from aqueous solution. Solution pH determines the nature of the charge (either positive or negative) on the surface of the bacteria, and thereby determines the nature of the force (repulsion or attraction) between the bacteria and the heavy metal ions.

ANOM technique for process parameter optimization
The significance of S/N ratio plotted in Figs 1-5 was as a tool for process parameter optimization. The optimum condition is measured at factor-level combination where the S/N ratio has the maximum value. Maximum values of S/N ratio were recorded to be 38.93 (Fig. 1), 39.17 (Fig. 2), 39.09 (Fig. 3), 39.20 (Fig. 4) and 38.97 (Fig. 5), where pH, temperature, time, inoculum concentration and Cd(II) initial concentration were 4, 303 K, 4 h, 15 v/v % and 50 mg/L, respectively. Thus, the factor-level combination that gives optimum Cd(II) removal from solution using bacterial isolates from sawdust was A2, B1, C4, D3 and E1.
A similar study where only 4 factors were considered presented similar results for optimum biosorption of copper and nickel from solution using Aspergillus sp. fungi (Pundir et al., 2018).
In order to confirm the efficacy of bacteria isolates from sawdust in removing Cd(II) from aqueous solution, a laboratory test was conducted at the predicted optimum condition (pH = 4, temperature = 303 K, time = 4 h, inoculum concentration = 15 v/v % and Cd(II) initial concentration = 50 mg/L) to calculate the percentage of heavy metal removal. At these parameter levels, the percentage of Cd(II) removed was 99.53%. This reveals the efficiency of the bacteria used to remove Cd from solution at optimum operating conditions. Table 7 compares the conditions at which optimum removal of Cd(II) was achieved in this study with results from the literature. Figure 6 represents the 3D surface plot showing effects of interaction of pH and temperature (Fig. 6a), pH and time (Fig. 6b), pH and inoculum concentration (Fig. 6c), pH and Cd(II) initial concentration (Fig. 6d), temperature and time (Fig. 6e), temperature and inoculum concentration (Fig. 6f), temperature and Cd(II) initial concentration (Fig. 6g), time and inoculum concentration (Fig. 6h), time and Cd(II) initial concentration (Fig. 6i), inoculum concentration and Cd(II) initial concentration (Fig. 6j), on Cd(II) biosorption from aqueous solution using bacterial isolates from sawdust. A high level of complementary interactive effects was exhibited among the investigated factors at different levels for the biosorption process. The plots were executed via plotting any two independent variables against each other while other variables were held constant. All actual factors were kept constant as Level 1, such that pH, temperature, time, inoculum concentration and Cd(II) initial concentration values were 2, 303 K, 1 hr, 5 v/v (%) and 50 mg/L, respectively. The percentage of Cd(II) removed from aqueous solution was greater than 76% in each of the plots. This affirms the high efficacy of bacterial isolates from sawdust as an effective biosorbent for Cd(II) removal from aqueous solution.

Fourier transform infrared spectroscopy (FTIR)
Figures 7a and 7b present the FTIR spectra of bacterial isolates from waste sawdust, before and after Cd(II) biosorption from aqueous solution. Presence of sharp peaks is a strong indication that active functional groups are present on the surface of bacterial isolates (Haq et al., 2015). A shift in sharp peaks is a strong indication that biosorption of the contaminant has taken place (Popoola, 2019b). Major sharp peaks observed at 3 583.41; 2 925.74 and 2 396.11 cm -1 before Cd(II) removal (Fig. 7a) shifted to 3 436.33; 2 929.5 and 2 355.43 cm -1 , respectively, after Cd(II) removal from aqueous solution (Fig. 7b). The major assignment at these respective wavelengths could be attributed to -OH stretching (Chojnacka et al., 2005), suggesting that this functional group is responsible for binding the metallic ions present in solution. Presence of the -OH group on the bacterial surface makes its surface negatively charged, which enhances the removal of positively charged Cd(II) from aqueous solution at a higher solution pH. This is a strong indication of bacteria isolates ability to take up Cd(II) from aqueous solution.

Scanning electron microscopy (SEM)
The SEM image of bacterial isolates from sawdust before (Fig. 8a) and after Cd(II) removal (Fig. 8b) are shown in Fig. 8. A porous morphological nature was shown before Cd(II) removal, which enhances the biosorption of the contaminant from aqueous solution. After the removal, the multiple layers of walls on the bacterial inner surface were seen to be covered with the Cd(II) ion, suggesting the effectiveness of the bacterial isolates as a biosorbent for Cd(II) in aqueous solution (Abou-Taleb et al. 2017). Table 8 presents the characterization of the bacterial isolates. The isolates were found to be motile when examined under the microscope. The isolates did not ferment glucose, sucrose, mannose and lactose and also exhibited gram-negative reaction attributes. They produce blue-green pigment and responded negatively to methyl red. However, the isolates responded positively to catalase, oxidase and citrate tests (Lennox et al., 2019;Hossain et al., 2013;.

Biosorption mechanism
Different mechanisms of heavy metal biosorption from aqueous solution have been proposed and developed (Wu et al., 2010). In this study, the proposed mechanism of Cd(II) removal from solution using bacterial isolates from sawdust includes electrostatic interaction, surface complexation and ion exchange.

Electrostatic interaction
The FTIR analysis has revealed the presence of -OH groups on the bacterial isolates' (BI) surface, with three sharp peaks revealed indicating -OH stretching. The cadmium salt is a bivalent positively charged heavy metal ion with a strong affinity for negatively charged surface biosorbent. At high pH, the surface of the bacteria is more negatively charged and, thus, loss of protons (deprotonation) occurs, with formation of water, as presented in Eq. 12.
Strong electrostatic forces then prevailed which enhanced the strong attraction between Cd(II) and bacterial isolates, as presented in Eq. 13.

Surface complexation
At low pH, the bacterial surface becomes more positively charged, and thus a gain of protons occurs (protonation), as presented in Eq. 14. As such, the protonated bacteria forms complexes at the cell surface.

Ion exchange
Under this mechanism, the counter ions (X + ) present in polysaccharides (a main constituent of bacterial isolates' cell wall) are exchanged with bivalent cadmium ions resulting from covalent bonding between the two, as presented in Eq. 16.

Other mechanisms
Other proposed mechanisms, as suggested by previous studies, could be interaction with oxygen-containing functional groups (Fawzy et al., 2019); precipitation interaction involving inorganic minerals such as carbonates, phosphates, and silicates (Huang et al., 2018); and bioaccumulation and pore surface physical agglomeration (Iqbal et al., 2009).

CONCLUSIONS
The results obtained from this present study revealed bacterial isolates from sawdust to be an effective biosorbent for the biosorption of Cd(II) from aqueous solution. Taguchi experimental design with L 16 4-level factors orthogonal array was utilized to investigate the optimal process parameters that would give maximum percentage removal of Cd(II) from aqueous solution. The confirmatory experiment conducted at the predicted optimal conditions (pH = 4, temperature = 303 K, time = 4 h, inoculum concentration =15 v/v % and Cd(II) initial concentration = 50 mg/L) revealed 99.53% removal for Cd(II). Analysis of variance revealed the order of factors' influence to be Cd(II) initial concentration (33.87%) > time (26.14%) > inoculum concentration (20.05%) > temperature (8.64%) > pH (6.07%). Effect of parameter interaction revealed the percentage of Cd(II) removed from aqueous solution to be greater than 76% in each of the 3D surface plots. Fourier transform infrared analysis revealed the presence of -OH on the bacterial surface as the main active functional group enhancing Cd(II) biosorption. Scanning electron microscopy revealed the presence of small cavities on the bacterial surface, with a porous inner multilayer. Mechanisms of biosorption were proposed to be electrostatic interaction, surface complexation and ion exchange. With reference to these observations, bacterial isolates from sawdust could effectively be applied as biosorbent for Cd(II) removal from aqueous solution.