Production of a bioflocculant by using activated sludge and its application in Pb(II) removal from aqueous solution

2.1 Activated sludge

Activated sludge samples were obtained from Sanwayao sewage treatment plant, Sichuan province, China. This sewage treatment plant treats approximately 50,000 m³ of domestic sewage per day by using Anaerobic-Anoxic-Oxic (A²/O) technology. Samples of sludge were collected from secondary sedimentation tanks. Prior to bioflocculant production, raw sludge samples were subjected to sterilization (ST), alkaline–thermal (ALT) and acid–thermal (ACT) treatments. Sterilization (steam sterilization) was performed 30 min at 121ºC according to [19]. ALT treatment was carried out by autoclaving at 121ºC for 30 min after adjusting the pH value of the sludge to 10 with 1 M sodium hydroxide (NaOH). In ACT treatment, the pH value of the sludge was adjusted to 2 using 1 M hydrochloric acid (HCl) prior to autoclaving for 30 min at 121ºC.

2.2 Bioflocculant production

The bioflocculant–producing strain used in the following experiments was Saccharomyces cerevisiae, purchased from the China Center for Type Culture Collection. To produce bioflocculant, the strain was inoculated in 100 mL of pre-treated activated sludge and incubated on a reciprocal shaker (SHA–A) at 150 rpm and 35ºC. After 60 h of cultivation, the fermentation broth with the flocculating components was obtained. The fermentation broth was centrifuged at 5000 rpm for 30 min to remove cells, and the supernatant with the flocculating components initially remained in this liquid form. Cold ethanol (4ºC) was added to the liquid at a volume ratio of 2:1. After precipitation for 24 h, the resulting precipitate was collected by centrifugation at 5000 rpm for 30 min. The pellet represented the crude bioflocculant [20]. The crude bioflocculant was dissolved in deionized water and purified by dialyzing overnight and was then lyophilized [21].

The total sugar of the bioflocculant was determined by the phenol–sulfuric acid method [22]. The protein of the bioflocculant was determined by the Bradford method [23]. The molecular weight of the bioflocculant was determined by gel permeation chromatography (GPC).

2.3 Pb(II) removal tests by the bioflocculant

Pb(NO₃)₂ (Hengxing Chemicals, China) was prepared by dilution of a 50 mg/L stock solution. Adsorption experiments were conducted to determine the effects of the bioflocculant dosage and solution pH in the presence of Ca²⁺ on the removal of Pb(II) from Pb(NO₃)₂ solutions. Before treatment, the pH of 300 mL of the above Pb(NO₃)₂ solution, as determined with a PHS-3C pH meter, was adjusted using 1 M NaOH or HCl. Subsequently, CaCl₂ and the bioflocculant (both prepared as 1 g/L solutions) were added into the beaker in turn. Next, the mixture was vigorously stirred (300 rpm) for 1 min and slowly stirred (80 rpm) for 4 min, and then allowed to stand for 10 min using a six–breaker jar tester. Finally, the supernatant was collected and the residual Pb(II) was determined. The removal efficiency (RE) and removal capacity of Pb(II) were calculated as follows:

Where C₀ and C_e were the initial and equilibrium Pb(II) concentrations (mg/L), respectively. V was the volume of the Pb(NO₃)₂ solution (L), and W_R was the weight of the bioflocculant used (g).

All of the measurements in this study were carried out in triplicate.

Ethical approval: The conducted research is not related to either human or animal use.

3.1 Bioflocculant production

Although a single sample of sludge was used to produce the input for bioflocculant production by a single strain, various pre-treatments of the sludge led to distinct bioflocculant yields. As seen in Figure 1, in this study, 4.45, 2.58 and 0.76 g of purified bioflocculant were harvested per L of fermentation broth of ALT, ST and ACT sludge, respectively. All of these yields from pre-treated sludge were higher than that harvested from raw sludge (0.12 g/L). The variations in bioflocculant yields from different fermented broths were attributed to the specific chemical/ physical changes induced by different treatments of the treated sludge [24].

Figure 1

Effects of sludge pre-treatment on bioflocculant yield. [Error bars refer to standard deviation (3 replicates for each treatment)].

Generally, treatment of sludge results in decomposition of organic components (including proteins and polysaccharides) of the material and the release of soluble carbon, and this soluble carbon is more suitable for utilization by microorganisms for bioflocculant production [25]. In this study, we found that bioflocculant yields following different pre-treatments were increased compared with that from the raw sludge, which might be explained by increases of soluble carbon levels that occurred regardless of the treatment method. The bioflocculant yield from the ALT sludge was the highest, meaning that the increase in soluble carbon likely was the highest in this case. Chen et al. [26] reported that under alkaline conditions, the soluble chemical oxygen demand and other low molecular weight soluble carbons were increased by sludge sterilization, and that these factors reached higher levels relative to acid treatment. Sun et al. [27] similarly concluded that sludge solubilization significantly increased with alkaline treatment and to a lesser extent by acid treatment. Aravinthan et al. [28] reported that mostly proteins of the sludge were solubilized by alkaline treatment whereas carbohydrate portions of the sludge preferentially solubilized by acid treatment. The carbon sources, nitrogenous organic materials, and nature of nutrients available in the medium change with the type of treatment and therefore can change bioflocculant secretion patterns and correspondingly the bioflocculant yields [29].

3.2 Characteristics of the bioflocculant

It has been reported that protein-dependent bioflocculants are sensitive to heat, while those whose active ingredient is polysaccharide are thermostable. These results might be explained by the fact that proteins can be denatured under hot conditions [25]. In the present work, the fermentation broth, which contains the flocculating components, from ALT-pretreated sludge was divided into 12 equal parts and separately treated at increasing temperatures over the range 10-120ºC for 30 min, after which the flocculating activities towards a kaolin suspension (4 g/L) were measured according to the method reported by [19].

Results (Table 1) showed that more than 90% of the flocculating activities of the fermentation broth were retained when the temperature was lower than 60ºC. However, when the temperature was adjusted to 90ºC, the flocculating activity declined to about 50%, which was further decreased to approximately 10% upon pretreatment at 120ºC (Table 1). The poor heat stability indicated that the bioflocculant produced when ALT-pretreated sludge is used as a precursor may have protein as a primary component. These results are consistent with those found by [19,25].

Chemical analyses showed that the protein content of the bioflocculant was 94.7% (w/w), while the polysaccharide content was 5.2% (w/w). The molecular weight of the bioflocculant was 4.24×10⁵ Da. Notably, other researchers have found that flocculant activity increases with increasing molecular weight [27].

3.3 Treatment of Pb(II) aqueous solution by the bioflocculant

Figure2 depicts the effects of bioflocculant dosage on the removal of Pb(II) without adjustment of the pH of the solution. In the presence of 50 mg/L CaCl₂, the removal efficiency of Pb(II) increased rapidly with increasing bioflocculant dosages from 0 to 6 mg/L. The maximum removal efficiency of 92.5% was obtained at the optimum bioflocculant dosage of 6 mg/L, and the corresponding removal capacity of Pb(II) by the bioflocculant was 77.1 mg/g. However, increasing the bioflocculant dosage above 6 mg/L had negligible effects on the increase in Pb(II) removal, which might be explained by the formation of aggregates at higher solid/liquid ratios or to precipitation of particles [4].

Figure 2

Effects of bioflocculant dosage on the removal of Pb(II) from aqueous solution. [Error bars refer to standard deviation (3 replicates for each treatment)].

To determine the role of CaCl₂ in Pb(II) removal by the bioflocculant, the effect of CaCl₂ was investigated by adding different mass fractions of CaCl₂ and 6 mg/L of the bioflocculant into 1L of 50 mg/L Pb(NO₃)₂ solutions without adjustment of the pH of the solutions. It can be seen from Figure3 that the removal efficiency of Pb(II) was relatively low (36.3%) without the addition of CaCl₂, but that the removal efficiency increased rapidly with increasing CaCl₂. The maximum value of 97.6% appeared when the CaCl₂ concentration was 70 mg/L, and the corresponding removal capacity of Pb(II) by the bioflocculant under this condition was 81.3 mg/g. Feng et al. [4] reported that the Ca²⁺ might increase the initial adsorption capacity of a bioflocculant by decreasing its negative charge. However, excessive Ca²⁺ (>70 mg/L) had negative effects on the Pb(II) removal efficiency, which may be due to competition between the Pb(II) and excess Ca²⁺ for the key functional groups of the bioflocculant such as –OH and –COO^- [5].

Figure 3

Effects of CaCl2 dosage on the removal of Pb(II) from aqueous solution. [Error bars refer to standard deviation (3 replicates for each treatment)].

The effects of solution pH value on the removal of Pb(II) by the bioflocculant were examined after 6 mg/L of bioflocculant and 70 mg/L of CaCl₂ were added into 1 L of Pb(NO₃)₂ solution. The pH values of the solutions were adjusted in the range of 2.5-7.5 to avoid the precipitation of Pb(II) in alkaline condition. As seen in Figure4, at pH 6.5, the removal efficiency of Pb(II) increased gradually and peaked at 98.5%, and at this pH, the corresponding removal capacity of Pb(II) by the bioflocculant was 50 mg/g. A similar optimal pH of 6 for Pb(II) removal has been reported by [5]. At low pH, the removal of Pb(II) was not optimal, likely because of competition for the functional groups of the bioflocculant between hydrogen ions (H⁺) and Pb(II) [30].

Figure 4

Effects of solution pH value on the removal of Pb(II) from aqueous solution. [Error bars refer to standard deviation (3 replicates for each treatment)].