Cadmium-containing Wastewater Induced by SRB: Effect of Multiple Ions and Mineralization Process

WANG Jiyong, ZHOU Jie, HE Wei, HUANG Pinyuan

(College of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan 430070, China)

Abstract: The effects of various general and heavy metal ions in acidic cadmium-containing wastewater on SBR desulfurization and cadmium removal under different concentrations were studied. and S2- inhibited SRB mineralization, and Mg2+, Ca2+, Fe2+, and Fe3+ promoted SRB mineralization at low concentration (＜50 mg/L). The inhibitory concentrations of Cu2+, Mn2+, Co2+, Zn2+, Pb2+, Hg2+, Cr3+, and Ni2+ were 10, 30, 2, 20, 25, 5, 30 mg/L, respectively. The inhibition order was Co2+＞Hg2+＞Cu2+＞Ni2+＞Zn2+＞Pb2+ ＞Mn2+＞Cr3+. Furthermore, the inverted microscopy and scanning electron microscopy (SEM) were used to observe the sediment crystallization process from macroscopic and microscopic viewpoints in the optimized ion environment. The experimental results show that under the mineralization of SRB, cadmium sediment crystallization is mainly divided into the rapid growth of bacteria, crystal nucleus production, block formation, and crystallization occuration. At the same time, X-ray diffraction (XRD) and energy-dispersive spectra (EDS) have also confirmed the sediment and crystallization.

Key words: SRB; mineralization process; cadmium; multiple ions

1 Introduction

In recent years, the rapid development of the economy and the improvement of people’s living standards pose a threat to the environment, especially heavy metal pollution has become a major obstacle to the aquatic environment[1,2]. As mining, metal smelting and electroplating industries directly discharge cadmium-containing acidic wastewater with high acidity, high Cd2+concentration, and high sulfate concentration, causing serious environmental pollution[3,4].

In the irrigation area contaminated by acid mine wastewater, water and soil are seriously damaged, soil acidification is serious; increased sulfate concentration will cause the imbalance of osmotic pressure of plant cell wall[5,6]. Cd2+is particularly harmful due to its ionic radius is similar to Ca2+[7], so that it is easier to lead to bioaccumulation in the food chain. Ultimately, that will harm the health of humans in the region[8,9]. Therefore, it is crucial to remove high concentrations of sulfate and heavy metal cadmium in wastewater to ensure that their presence in the environment is below the standard[10-12].

Traditional physical and chemical methods such as ion exchange, adsorption sediment, and electrochemistry have made some progress in treating wastewater[13,14]. However, traditional methods have some problems such as high cost, waste of energy, and generating secondary pollution[15]. Microbial remediation technology is a promising and practically effective technology as its environmentally friendly, low cost, and no secondary pollution[16,17]. At the same time, some microorganisms in heavy metal mining areas have also been reported to be involved in the fixed mineralization of heavy metal ions[18,19]. De J et al screened microorganisms with good heavy metal resistance, which can convert free heavy metals into very stable minerals, such as sulfides[20].

Many researchers are mainly based on the use of SRB to treat cadmium-containing acidic wastewater. SRB is one of the most effective strains for the treatment of cadmium-containing acidic wastewater[21-24]. The main mechanism of action is the use of sulfate as a terminal electron acceptor, a simple organic or inorganic compound as an electron donor, the conversion of sulfate in the environment to sulfur ions[25,26], and the rapid binding of heavy metal ions to immobilize heavy metal ions, which also has a very pronounced effect at low initial concentrations[27]. Gopi Kirana M et al used SRB to remove various heavy metals such as Cd(II) and Cu(II), and the obtained metal sulfides were precipitated in a crystal form, indicating that metal precipitate formation was related to the outer and inner cell surface of the SRB[28].

Studying the microbial mineralization process is more conducive to an understanding of the mineralization mechanism. Aleksei V Rusakov et al analyzed the effects of fungi and bacteria on the surface of different calcium-containing minerals, indicating that microbial metabolic activities affect the ore-forming morphology and crystallization process[29]. Chen Z et al immobilized Pb(II) with Bacillus cereus 12-2, suggesting that from amorphous sediment to crystalline minerals occur in cells, and enzyme-mediated mineral conversion[30].

However, it can be found from the literature that most of the research mainly analyzes the experimental conditions[31,32]or enzymes of different microbial mineralization by XRD or TEM, which mainly focus on biomineralization of Urease-producing bacteria[33,34], Besides, there are few studies on the mineralization process under microbial activity, and SRB is rarely reported.

In this study, we selected a strain of sulfate-reducing bacteria separated from acidic industrial wastewater with superior acid resistance, then studied the sediment of cadmium ions in a complex ions environment and the sediment morphology of the crystallization process. This article focused on the following aspects: cadmium ion sediment in different concentrations of general and heavy metal ions; SRB-induced evolution of Cd(II) mineralization morphology over time; mineralized sediment analysis of rough surface and its components; potential mechanism of mineralization and crystallization. We hope that this study can provide important information for SRB biomineralization characterization and mechanism research.

2 Experimental

2.1 SRB source and culture conditions

The SRB strain used in this study was isolated from a flower bed from Wuhan University of Technology, Wuhan, China. SRB was finally obtained by continuously screening and purifying Desulfovibrio species in the biomass[35].

Heterotrophic medium used in SRB culture was modified postgate liquid medium that contained 0.5 g/L KH2PO4, 0.05 g/L MgCl2·6H2O, 1.1 g/L Na2SO4, 0.05 g/L CaCl2, 1.0 g/L NaCl, 1.0 g/L NH4Cl, 2 g/L sodium lactate, and 0.5 g/L yeast powder with the initial pH 6.5 adjusted by 1 mol/L HCl or NaOH. The liquid medium was autoclaved at 121 ℃ for 20 min. After the medium cooled down in the sterilization and purification workbench, L-cysteine 0.3 g/L, ascorbic acid 0.3 g/L were added to it before ultraviolet sterilization. All chemicals and reagents used in this study were of analytical grade.

SRB was inoculated in the medium in an amount of 1% with 150 mL of the liquid medium, cultured in an anaerobic flask (250 mL), and the above procedure was repeated to obtain an SRB-enriched culture, which was carried out under aseptic conditions.

2.2 Effect of various general ions and heavy metal ions on SRB

There are usually many ions in the wastewater, which may affect the osmotic pressure on both sides of the cell wall[5], or may occupy the ion transport channels of the microorganisms[36], or may have toxic effects on the cells, thereby affecting the survival and metabolism of the microorganisms. To study the effect of different ions on the removal of Cd(II) by SRB, this study selected general and heavy metal ions as single-factor experiments in Table 1.

Table 1 Coexisting ion single factor experimental design

Table 2 Effect of general ions on cadmium removal rate of SRB

The medium to which S2-was added was only considered to be precipitated with Cd2+, so only the removal rate of SO42-was considered. When studying the influence of heavy metal ions, the total heavy metal concentration in the medium (including Cd2+) is 30 mg/L. The liquid sample was taken periodically during the experiment to confirm the change of the cadmium ion concentration and the sulfate concentration of the sample.

2.3 Degree of biomineralization of Cd(II) at different time

In order to study the mineralization and crystallization process of CdS under the mineralization of SRB, the initial concentration of the Cd(II) was determined at 25 mg/L for the research of the crystal morphology changes of different periods, in which concentration the bacteria can still grow up well meanwhile we can obtain enough sediment. SRB was cultured in a modified Postgate liquid medium as described previously with the initial pH 5.5, and the flasks were transferred to an orbital shaker at a temperature of 30 ℃ and a stirring speed of 90 rpm. A medium without SRB was used as a blank control. Liquid samples were taken periodically during the experiment to determine pH, cadmium ion concentration, and sulfate concentration; the precipitate was characterized at 18 h, 36 h, 48 h, and 60 h.

2.4 Related characterization of the metal bio-precipitates

Characterization of mineralized crystals includes scanning electron microscope (SEM), X-ray diffraction (XRD), energy-dispersive spectra(EDS), and inverted biological microscopy. The mineralized crystals obtained by SRB were centrifuged (8 000×g) for 5 min, washed twice with distilled water. The mineralized crystals after washing were divided into two groups. One group was re-added to distilled water and ultrasonically oscillated (50 kHz, 120 W) at 60 ℃ water bath for 20 min, hot centrifuged (8 000×g) for 5 min, washed twice with distilled water, then reserve it; another set was directly reserved. The above two groups of precipitates and crystals were freeze-dried and stored at 4 ℃.

SEM was used to characterize surface morphological changes in mineralized sediment during biomineralization and crystallization. The crystallization and main components were observed by XRD. The elemental composition was characterized by EDS, and the macroscopic changes of sediment crystallization during mineralization were observed by inverted biomicroscopy.

2.5 Analytical methods

The concentration of Cd(II) in the sample was determined by atomic absorption spectrometry(Thermo, ICE 3000 Series). The SO42-concentration of the water sample was measured by barium sulfate turbidimetry[37]. A biological microscope (37XE-PC, Shanghai Optical Instrument Factory No.1) observed the crystallization process. The content of polysaccharides was determined by the phenol sulfuric acid method[38], and the protein content was determined by coomassie bright blue dye-binding method. All experiments were performed in triplicate and expressed as means with standard deviation.

3 Results and discussion

3.1 Effect of general and heavy metal ions on desulfurization and removal of Cd (II)

The desulfurization rate of SRB under the influence of general ions and different concentrations is shown in Fig.1. The effects of ion species and concentration on the desulfurization rate of SRB are also different. The difference in ion species and concentration is significant for the desulfurization rate of SRB. The addition of CrO42-and S2-has an inhibitory effect on desulfurization, and the higher the concentration is, the more severe the inhibition is. When CrO42-is increased from 0 mg/L to 200 mg/L, the desulfurization rate decreases from 54.1% to 25.3% because the structureoccupying the transport channel of, which hinders the cell transportation. process and thus affects the desulfurization rate. Sulfide is a product of SRB action, which is toxic to SRB[39]. When the concentration of S2-increases from 0 mg/L to 200 mg/L, the desulfurization rate decreases from 54.8 % to 25.5%.

Fig.1 Effect of different ions and concentrations on desulfurization rate of SRB

Adding Mg2+, Ca2+, Fe2+, and Fe3+can promote desulfurization at low concentrations (＜50 mg/L) (the desulfurization rate can be increased by about 3%), but cause a certain inhibition at high concentrations (＞50 mg/L) (desulfurization rate is reduced by 2% to 6%). This is because Mg2+, Ca2+, Fe2+, and Fe3+are constituent elements of some enzymes (cytochromes, catalase, etc) or cell structures (ribosomes, cell membranes, etc) in SRB cells[40]. It promotes the growth of SRB within a suitable concentration range. Fe2+and Fe3+can also bind to S2-to reduce their inhibition of SRB. When the ion concentration is high, the osmotic pressure of the solution changes, which affects the process of material transportation. Therefore, the high concentration will have an inhibitory effect.

The cadmium removal rate of SRB under the influence of general ions and different concentrations is shown in Table 2. Different ions have different sediment concentrations, and they have different effects on the cadmium removal process of SRB. Mg2+and Ca2+do not affect the removal rate of cadmium. With the increase of CrO42-, Fe2+and Fe3+concentration, the removal rate of cadmium decreases. When the concentration is 200 mg/L, the cadmium removal rate decreases to 41.3%, 43.3%, and 38.6 % which is reduced by more than half.The principle of suppression is mainly as follows. CrO42-hindered the transport of SO42-,which reduced the production of S2-and affected the removal of cadmium. Fe2+and Fe3+combined with S2-reduced the sediment of Cd2+, thus generating iron sulfide is detrimental to cadmium removal.

Table 3 Effect of heavy metal ions on cadmium removal rate of SRB

By taking into account the desulfurization and cadmium removal, Mg2+, Ca2+, Fe2+, and Fe3+promote desulfurization at low concentrations (≤50 mg/L) without affecting cadmium removal. However, it was affected at high concentrations (＞50 mg/L). While the effect of CrO42-ion inhibiting desulfurization and removal of cadmium becomes more and more serious with increasing concentration. Therefore, in the SRB optimized culture study, an appropriate amount of Mg2+, Ca2+, Fe2+, and Fe3+can be added to enhance the desulfurization and cadmium removal ability of SRB.

The effect of heavy metal ion species and concentration on the desulfurization rate of SRB is shown in Fig.2. Heavy metals affect the activity of SRB, respiratory metabolism, and substance synthesis and are toxic to SRB, while SRB is also tolerant to heavy metals[41]. It can be seen from the figure that different heavy metals have an inhibitory effect on the desulfurization of SRB. Different heavy metal ions show inhibition of desulfurization of SRB but differ in extent. From the point of view of the decrease of desulfurization rate, when the Cd2+was chosen as 30 mg/L, the desulfurization rate is above 54%. This is because SRB is obtained by sieving at high cadmium ion concentration and is highly resistant to cadmium. It also confirmed that the strain can improve its resistance and tolerance by adapting itself to the environment in a specific environment.

Fig.2 Effect of different heavy metal ions and concentration on desulfurization rate of SRB

Although SRB can remove most heavy metal ions by metabolism, heavy metal ions generally inhibit SRB, and the higher the concentration is, the greater the inhibition is. However, by domesticating SRB, it can withstand higher concentrations, so that it can play a greater role in the treatment of heavy metal wastewater.

The cadmium removal rate of SRB under heavy metal ions and different concentrations are shown in Table 3. Because S2-can also form precipitates in combination with different heavy metal ions and they can also affect the removal of Cd2+by affecting SRB. In order to make the heavy metal concentration in the suitable growth range, the sum of the added heavy metal ion and the Cd2+is 30 mg/L. When the other ion is 2 mg/L, the cadmium rate can reach more than 90%. With the increase of heavy metal ion concentration (＜20 mg/L), Cu2+, Co2+, and Hg2+have a great influence on cadmium removal rate, and Mn2+, Zn2+, Pb2+, Cr3+, and Ni2+have less influence. When the concentration of other heavy metals increased to 25 mg/L (Cd2+was 5 mg/L), the cadmium removal rate in the presence of Cu2+, Co2+, and Hg2+is still very low, because the desulfurization rate was also low and the solubility product of HgS is smaller than CdS.

3.2 Characterization of the crystallization pr ocess

The reduction of sulfate and Cd2+, as well as the formation of metal mineral sediment and crystallization, proves the key factors of desulfurization, cadmium removal efficiency, mineralization and crystallization process[21]. The characterization of mineralized crystals includes SEM, XRD, EDS, and other analyses to understand the changes in the crystalline composition of the SRB during the study and its elemental composition. A clear SEM image of mineralization by SRB is shown in Fig.3, which mainly forms irregular massive metal precipitates. The surface of the metal precipitate exhibits uneven roughness, while a large amount of crystalline minerals is present on the surface. To highlight the influence of Cd elements in the crystallization process, trace amounts of Mg2+, Fe2+, and Fe3+will not be considered in EDS.

Fig.3 (a) SEM image of mineralized crystalline morphology; (b)XRD pattern of mineralization and crystallization; (c) EDS spectrum from a spot on the mineralization and crystallization

From the EDS results, the atomic ratio of Cd and S is close to 2:1, indicating that the formed mineralized precipitate is precipitated in the form of CdS, and other forms of sediment, such as Cd(OH)2. From the XRD pattern and its comparison with the standard XRD card, there are more CdS crystals with better crystallinity in the precipitate. It is speculated that the surface roughness of metal precipitates is related to the growth and metabolism of bacteria to produce extracellular polymer (EPS). The surface is mainly soluble polysaccharides and proteins. Agglomerate adsorption of EPS also affects the formation of the heavy metal precipitate. The experimental results of the study are similar to Zinkevich V et al[42].

Take the supernatant of the solution after sonication and centrifugation, we found that the mass ratio of polysaccharide to protein in this solution is about 9:1. By ultrasonic treatment, the condition of the precipitated surface was changed from Fig.4(a) to Fig.4(b), and the surface was apparently smooth. This may be due to the influence of ultrasound, which caused the adsorbed polysaccharide and protein to be separated from the surface, and the roughness was reduced.

Fig.4 (a) The control group; (b) The ultrasound treatment group

This article also studies the process of metal crystallization and sediment during mineralization in detail. Fig.5 shows that under an inverted biomicroscope, the sediment begins to occur, from small to large. At 18 h, only the white SRB cell suspension can be seen. At this time, the early stage inverted microscope intuitively recorded SRB-induced Cd2+sediment crystallization, and Fig.6 is a sediment crystallization process under SEM from a small perspective. At 18 h, it can be seen that a large number of spherical SRB cells are clustered together, indicating that SRB is rapidly growing and reproducing at this time. At 36 h, many small massive precipitates were attached around the SRB cells. The cells began to agglomerate and crystallize. At 48 h, the cell structure was almost invisible, and small particles precipitated to form a large precipitate. At 60 h, a large block precipitate formed and a smaller crystal structure began to appear.

Fig.5 SRB induced Cd2+ sediment process under an inverted biological microscope: (a) (×25), (b) (×25), (c)(×40), and (d) (×40) are the sediment liquids collected at 18, 36, 48, and 60 h, respectively

Fig.6 SRB induced sediment of Cd2+ in (a) 18 h, (b) 36 h, (c) 48 h, (d) 60 h by SEM

That the SRB induces Cd2+sediment and crystallization contains four processes: bacterial growth and reproduction; generation of small precipitated particles, crystallization with cells as crystal nucleus; formation of large precipitates; crystal structure on the precipitated surface.

4 Conclusions

In this article, the effects of various general and heavy metal ions of different concentration conditions on the desulfurization and removal of cadmium by SRB were investigated. Generally, general and heavy metal ions show inhibition of SRB with increasing concentration, but the degree of inhibition is quite different. In the process of cadmium mineralization and crystallization induced by SRB, the sediment crystallization process was observed from macroscopic and microscopic using an inverted microscope and scanning electron microscope. The results showed that cadmium sediment crystallization was mainly divided into the rapid growth of bacteria, produced, a massive precipitate formed, crystals appeared. Moreover, it was further confirmed that the ratio of polysaccharide to protein in the precipitated surface rough polymer was close to 9:1, which was similar to the EPS composition. It can be seen from EDS that the proportion of CdS crystals in mineralized sediment is small, increasing the crystal size, and converting cadmium into a more stable binder phase is the future research direction.