Microbial flocculant produced by a novel Paenibacillus sp.,strain A9,using food processing wastewater to replace fermentation medium and its application for the removal of Pb(II) from aqueous solution

Abstract

Due to high production costs, the popularization and application of microbial flocculants in the field of water treatment have been limited. In this study, the capture of lead ions by the fermentation broth of a novel Paenibacillus sp. strain A9 and cultured with food wastewater was further investigated. The results revealed that the production of MBFA9 could be increased significantly by adding a small amount of carbon and nitrogen to food wastewater. Under the best experimental conditions (pH 8.5, culture temperature 30°C, 150 r/min), adding 1% (m/v) carbon and 0.1% (m/v) nitrogen to 1% (v/v) wastewater resulted in a yield of MBFA9 of 6.29 g/l. At a temperature of 30°C, pH of 5, contact time of 35 min, and FBA9 dosage of 5%, the removal rate and removal capacity of Pb(II) reached the highest values of 95.1% and 317 mg/g, respectively. Field emission scanning electron microscopy analysis indicated that bacterial cells, metabolite small molecule acids, and MBFA9 in FBA9 all contributed to the removal of Pb(II). Fourier-transform infrared spectrometry analysis indicated that functional groups such as –OH, –COOH, –CO, and –NH₂ existed in MBFA9 and on the cell surface. Various mechanisms involved in Pb(II) removal can occur simultaneously, including cell surface adsorption, microcrystallization, and biological flocculation.

Keywords

Adsorption bioflocculant removal fermentation broth MBFA9

Introduction

With the development of industrial production, the amount of heavy metals discharged to the environment is increasing, resulting in different degrees of water pollution (Ao and Guan, 2018; Zhang et al., 2019). A large amount of acid wastewater containing lead is discharged by the electroplating, pigment, rubber, and fuel production processes. Lead is a toxic heavy metal that threatens the natural environment and can also enter the human body through the food chain, skin, digestive tract, and respiratory tract, after which it causes damage to organs, especially in children (Guo, 2015; Thouria et al., 2018; Wang et al., 2018; Zhou et al., 2017).

Chemical precipitation, electrolysis, adsorption, and ion exchange are methods commonly used to control heavy metal pollutants. The removal of heavy metals by microbial methods has many advantages, such as low cost, simple operation, strong selectivity, and good treatment effect. Many studies have been conducted to investigate heavy metal wastewater treatment, and adsorption technology has recently received a great deal of attention because of its advantages of high efficiency, low cost, and safety (Nharingo et al., 2015; Xia et al., 2018; Xue et al., 2018). Some scholars pay close attention to the use of the adsorbance ability of metabolites produced by microbial fermentation, such as microbial flocculants (MBFs), to remove heavy metals. For example, Yao et al. (2013) used MBF produced by the flocculant-producing bacteria Bacillus mucilaginosus to treat wastewater containing Fe(III) and Pb(II) and introduced CO₂ to enhance the removal effect. They found that MBF has an adsorption effect on metal ions and can also be used as a nucleating medium for the formation of carbonate minerals via reaction with CO₂. Guo and Yu (2014) used the microbial flocculant MBFR10543 to treat copper-containing wastewater and found that the wastewater treatment rate reached 96.9%, and its adsorption mechanism was electric neutralization and adsorption bridging. Many studies have also investigated the adsorption of cadmium, lead, chromium, and other heavy metals by microbial cells. Zeng et al. (2008) believed that some groups containing nitrogen and oxygen on the surface of Bacillus cereus HQ-1 cells could complex Ag⁺, and thus adsorb Ag⁺.

Many studies of the removal of heavy metals by microbial methods have focused on one mechanism, such as the purification product MBF produced by microbial metabolism, or the adsorption function of special bacteria/flora. However, there may be several mechanisms involved in the adsorption of metal ions at the same time, such as bacterial species, MBF, or small molecule metabolism obtained by fermentation of bacteria. These products and other components may contribute to the removal of heavy metals to varying degrees. If functional strains can be cultured by directional fermentation and heavy metals can be removed by fermentation broth, the tedious process of MBF purification or cell collection can be omitted, and the cost of heavy metals removal can be greatly reduced.

Because of the high production cost of MBFs, its popularization and application are limited. Therefore, determining how to use inexpensive raw materials to replace carbon and nitrogen sources in culture medium or organic wastewater to replace flocculant-producing bacteria culture media to reduce the production costs of MBFs and develop new culture media has become the main research goal and direction of researchers.

Some researchers use brewery wastewater, brewing sauce wastewater, or cassava starch wastewater instead of culture medium for microbial growth and flocculant production, which greatly reduces the cost of microbial culture while enabling the reuse of waste resources. Therefore, this study investigated the use of food processing wastewater instead of carbon and nitrogen sources in fermentation medium for flocculant-producing bacteria, and the effect factors and mechanism of removal of heavy metal lead ions in water were studied. The results presented herein lay a foundation for maximizing the yield of MBFs and its large-scale application in industrial production.

Materials and methods

Reagents

Pb(NO₃)₂ (Sinopharm Chemical Reagent Co., Ltd, China) was prepared by dilution of 1 g/l stock solution, and fresh diluents were used in each experiment. NaOH and HCl (Sinopharm Chemical Reagent Co., Ltd, China) were prepared at a concentration of 1.0 mol/l. Unless otherwise stated, all reagents used were analytically pure.

Source of strains and wastewater

The MBF-producing bacteria Paenibacillus sp. strain A9 used in this experiment was isolated from peach tree cultivation soil (10 cm depth) in Liaoning Province, China. The flocculability of fermentation broth A9 (FBA9) to kaolin solution could reach 96%. Paenibacillus sp. strain A9 was identified by sequence analysis, physical and chemical properties, DNA hybridization, and fatty acid detection as a new strain of Paenibacillus sp., which was named Paenibacillus shenyangensis A9^T and preserved in the Preservation Center of the Institute of Microbiology, Chinese Academy of Sciences (Preservation No. CGMCC2040).

Food processing wastewater (abbreviated as wastewater) was taken from a food processing company in Shenyang. The main products of the wastewater were feed with lysine and food freshener nucleotides, as well as corn protein feed, corn protein powder, corn gluten feed, sugar meal, and corn germ. The chemical properties of the food wastewater (COD, NH₄⁺–N, NO₃⁻N, NO₂⁻–N, Organic-N, TN, and pH) were determined in accordance with the Standard Methods.

Culture media

Fermentation medium A: A single colony was inoculated into 100 ml medium consisting of 20.0 g glucose, 5.0 g yeast extract, 2.0 g KH₂PO₄, 5.0 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, and 0.1 g NaCl in 1 l distilled deionized water (initial pH 7.2–7.5) that had been autoclaved at 121°C for 20 min.

Wastewater culture medium B: A single colony was inoculated into 100 ml medium consisting of 1% (v/v) wastewater, 2.0 g KH₂PO₄, 5.0 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, and 0.1 g NaCl in 1 l distilled deionized water (initial pH 7.2–7.5) that had been autoclaved at 121°C for 20 min.

Wastewater enrichment medium C: A single colony was inoculated into 100 ml medium consisting of 1% (v/v) wastewater, 10.0 g starch, 1.0 g yeast extract, 2.0 g KH₂PO₄, 5.0 g K₂HPO₄, 0.2 g MgSO₄·7H₂O, and 0.1 g NaCl in 1 l distilled deionized water (initial pH 7.2–7.5) that had been subjected to high pressure sterilization at 121°C for 20 min.

Analysis of FBA9 component

Activated bacteria A9 was inoculated in wastewater enrichment medium C at a 3% inoculation volume with OD₅₅₀ between 0.01 and 0.02. FBA9 was obtained by incubating the samples at 30°C and 150 r/min for 48 h. Small molecular organic acids in FBA9 were detected by liquid chromatography–mass spectrometry (LC/MS) (6530-UHD, Agilent, US), and polysaccharide MBF produced by strain A9 (MBFA9) was determined by the anthrone-sulfuric acid method.

LC/MS column: Agilent, 100 mm × 2.1 mm, 1.8 µm; column temperature: 40°C; flow rate: 0.35 ml/min; mobile phase A: water + 0.1% formic acid; mobile phase B: acetonitrile + 0.1% formic acid; injection volume: 4 µl; automatic injector temperature: 4°C.

Anthrone-sulfuric acid method: Briefly, 1 ml of distilled water and 8 ml of anthrone reagent (0.2 g anthrone and 100 ml concentrated sulfuric acid) were added to a test tube, then treated with boiling water and ice water for 10 min, respectively. Samples were subsequently allowed to stand for 10 min at room temperature, after which the absorbance at 620 nm was determined by spectrophotometry. A standard curve was determined using glucose as the standard solution and a blank solution as a reference.

Flocculant activity test and MBFA9 preparation

Kaolin (0.5 g) was weighed in a 100 ml graduated cylinder, after which 100 ml of distilled water was added, samples were shaken well, and 0.1 ml FBA9 was added. After repeated shaking, the samples were allowed to sit for 10 min. Next, OD₅₅₀ of 50 ml of supernatant was determined by ultraviolet spectrophotometry. The system without FBA9 under the same conditions was used as a blank control. The formula for calculating flocculation activity that expressed the flocculation rate (FR) was as follows

FR(%)= \frac{A-B}{A} ×100%

(1)

where A is the optical density of kaolin suspension without FBA9 at 550 nm, and B is the optical density of kaolin suspension with FBA9 at 550 nm.

The FBA9 was diluted three times and then centrifuged at 8000 r/min for 15 min to remove the bacteria, after which it was concentrated in a rotary evaporator, precipitated with two volumes of anhydrous ethanol, and held at 4°C for 24 h. After mixing, filtering, and collecting the precipitate, the aforementioned operations were repeated with one volume of anhydrous ethanol. The white flocculants were collected twice and frozen for 24 h in an ultra-low temperature refrigerator, then lyophilized into dry powder MBFA9 using a freeze dryer.

Determination of Pb(II) removal rate (RR)

After heating in a water bath at 70°C for 30 min and autoclave sterilization for 30 min, different amounts of FBA9 were added to 10 ml Pb(II) solution and adjusted to different pHs, temperatures, and contact time for the reaction. Each container was then centrifuged for 10 min at 6000 r/min. The concentrations of Pb(II) in the supernatant were then determined using a Prodigy XP ICP. The RR of Pb(II) was calculated as follows

RR (%)= \frac{C_{0} {-C}_{e}}{C_{0}} × 100 %

(2)

where C₀ and C_e (mg/l) are the initial and equilibrium Pb(II) concentrations, respectively.

Investigation of factors that affect the Pb(II) RR

To investigate the adsorption behavior in different pH values, 5% (v/v) FBA9 was added into 100.0 ml of solution containing 50.0 mg/l Pb(II) for contacting 35 min at 30°C. Next, 1.0 mol/l HCl and NaOH solution were used to adjust the desired solution pH values from 2.0 to 6.0. Finally, the supernatant was used to determine the Pb(II) concentration by ICP. The effects of FBA9 dosage on the Pb(II) RR were studied using FBA9 at doses ranging from 0.5 to 10.0% (v/v) in 50.0 mg/l Pb(II) solution (100.0 ml) with a pH value of 5.0 and a contact time of 35 min at 30°C. The effects of contact time on the Pb(II) RR were studied using 5% (v/v) FBA9 in 50.0 mg/l Pb(II) solution (100.0 ml) with a pH of 5.0 and a contact time of 5–40 min at 30°C. The effects of temperature on the Pb(II) RR were determined using 5% (v/v) FBA9 in 50.0 mg/l Pb(II) solution (100.0 ml) with a pH value of 5.0 and a contact time of 35 min at 15–45°C.

Fourier-transform infrared spectrometry (FTIR) analysis of MBFA9

To investigate the characteristics and mechanisms of the removal process, FTIR was employed to examine the interactions between Pb(II) and functional groups on the fermented products.

The FBA9 was diluted three times with distilled water and then centrifuged for 15 min at a speed of 8000 r/min. After evaporation and concentration by rotary evaporation, FBA9 was precipitated by addition of ethanol in three rounds, stored at 4°C for 24 h, and washed with 95% ethanol. Then MBFA9 was collected for Pb(II) removal under conditions of 50.0 mg/l Pb(II) solution (100.0 ml), 5% FBA9 dosage, temperature of 30°C, pH of 5, and contact time of 35 min. MBFA9 and MBFA9-Pb(II) before and after Pb(II) removal were used for FTIR detection.

A mass of dry MBFA9 was mixed with dry finely powdered potassium bromide in a ratio of 1:100. The mixture was spread uniformly in a suitable disk, then subjected to a pressure of 800 MPa, after which the spectra were recorded in the range of 400–4000 cm⁻¹ using a Nicolet 380 Thermo Scientific Fourier transform infrared spectrometer.

Field emission scanning electron microscopy (FESEM) analysis of FBA9

The surface ultrastructure of original and Pb(II)-loaded FBA9 was analyzed by FESEM. After Pb(II) removal under the conditions of 50.0 mg/l Pb(II) solution (100.0 ml), 5% FBA9, 30°C, pH 5, and a contact time of 35 min, FBA9 and FBA9-Pb(II) were centrifuged for 5 min at 8000 r/min. Collected bacteria and sediments were cleaned by phosphate buffer and ethanol gradient dehydration and dried by vacuum drying. Then dried samples were sputter-coated with gold and scanned with FESEM in a low-vacuum mode at an accelerating potential of 20 kV.

Results and discussion

Substitution of food wastewater for fermentation medium

The main components of wastewater used in this study were corn starch, amino acids, and nucleotides. The chemical indexes of the fresh wastewater were determined and are presented in Table 1.

Table 1.

Chemical properties of food wastewater.

pH	NH₄⁺–N (mg/l)	NO₃^—N (mg/l)	NO₂^—N (mg/l)	Organic-N (mg/l)	TN (mg/l)	COD (mg/l)
5.44	60,972.97	2223.68	296.35	52,807.00	116,300.00	247,166.67

COD: Chemical oxygen demand; TN: Total nitrogen.

The organic nitrogen and COD of the food wastewater were very high, which ensures adequate carbon and nitrogen sources for the growth of microorganisms. In addition, the pH value was 5.44, which would meet the growth needs of most microorganisms.

FBA9 was prepared by inoculation of A9 (OD₅₅₀ between 0.01 and 0.02) into fermentation medium A, wastewater medium B, and wastewater enrichment medium C, respectively (3% inoculation), under culture conditions of pH 7.2–7.5 and 150 r/min at 30°C for 48 h. The FR of FBA9 and MBFA9 yield under different culture conditions is shown in Table 2.

Table 2.

Comparison of flocculation rate and MBFA9 yield of FBA9 in three culture mediums.

Culture medium	Flocculation rate (%)	MBFA9 yield (g/l)
A	85.21 ± 2.73	4.60 ± 0.19
B	37.56 ± 4.16	0.21 ± 0.04
C	87.64 ± 2.55	6.29 ± 0.26

MBFA9: MBF produced by strain A9.

The FR of FBA9 and MBFA9 yield with wastewater enrichment medium reached 87.64% and 6.29 g/l, respectively, which were superior to those of wastewater medium and fermentation medium. Although A9 could grow well in wastewater medium, the yield of MBFA9 and FR was very low. However, the yield of MBFA9 was increased significantly in response to the addition of a small amount of carbon and nitrogen to the wastewater medium.

Food wastewater contains not only abundant nutrients but also metal ions, amino acids, organic acids, and vitamins. These complex components may promote or inhibit the growth of bacteria. Different trace elements have an impact on the growth and diversity of microorganisms, and the concentration of trace elements has different effects on microorganisms (Vahjen et al., 2011). Ali et al. (2012) found that when the concentration of Zn²⁺ was adjusted from 50 to 200 mg/l, the growth of Bacillus spp. increased gradually, then decreased. Specific concentrations of iron ions inhibited microbial growth, while other trace elements, such as excessive iodine, inhibited bacterial growth and reduced microbial abundance (Zhao et al., 2013). Therefore, it is necessary to control the concentration of food wastewater to provide adequate nutrients, as well as to control the toxic side effects to make the microbial growth environment more suitable.

Components of FBA9

There were A9 cells, small molecular organic acids, and MBFA9 in FBA9. The main constituents and groups of FBA9 are shown in Table 3.

Table 3.

Constituents and groups of FBA9.

FBA9	Constituents	Groups
Bacteria cells	Peptidoglycan, protein, lipid	–COOH, –OH, –CO–, –NH₂, –SH, –CONH–, –PO₃H₂, imidazole groups
Small molecular organic acids	Malic acid, succinic acid, citric acid	–COOH, –OH, –CO–, –NH₂
MBFA9	Mannose, glucose, acidic polysaccharides, acetaminopolysaccharides	–COOH, –OH, –CO–, –NH₂

FBA9: fermentation broth A9; MBFA9: MBF produced by strain A9.

Investigating factors that affect RR and removal capacity of Pb(II)

The solution pH is a very important parameter that affects the heavy metal adsorption process. The effects of the initial solution pH on Pb(II) removal by FBA9 are shown in Figure 1(a). The solution pH values were adjusted between 2.0 and 6.0. The hydrolysis constants (log k1 = 6.48, log k2 = 11.16, and log k3 = 14.16) of Pb(II) suggest that the Pb(II) species were present in different proportions including Pb²⁺, Pb(OH)⁺, Pb(OH)₂⁰, and Pb(OH)₃⁻ at different pH values (Xiang et al., 2017). The existence of Pb(II) in solution varies with pH, dissolution equilibrium, and complexation equilibrium of Pb(II) and is shown as follows

P b^{2 +} + 2 O H^{-} \to Pb {(OH)}_{2} (s) \to P b^{2 +} + 2 O H^{-}

P b^{2 +} + O H^{-} = Pb {(OH)}^{+}

P b^{2 +} + 2 O H^{-} = Pb {(OH)}_{2}

P b^{2 +} + 3 O H^{-} = Pb {(OH)}_{3}^{-}

Figure 1.

(a) Effect of solution pH on Pb(II) RR and removal capacity of FBA9 (the FBA9 dosage=5% (v/v), Pb(II)=50.0 mg/l, treatment time: 35 min, temperature: 30°C); (b) effect of FBA9 dosage on Pb(II) RR and removal capacity of FBA9 (pH = 5.0, Pb(II)=50.0 mg/l, treatment time: 35 min, temperature: 30°C); (c) effect of contact time on Pb(II) RR and removal capacity of FBA9 (pH = 5.0, the FBA9 dosage=5% (v/v), Pb(II)=50.0 mg/l^, temperature: 30°C); (d) effect of temperature on Pb(II) RR and removal capacity of FBA9 (pH = 5.0, FBA9 dosage=5% (v/v), Pb(II)=50.0 mg/l, treatment time: 35 min).

Normally, at pH < 7, Pb²⁺ is present in the solution, while in the pH range of 7 – 9, Pb(OH)⁺ is the predominant form of lead in the solution. When the pH of a solution is higher than 8, Pb(OH)₂⁰ starts to appear, and its content increases with increasing solution pH, while at > pH 9, Pb(OH)₃⁻ appears and Pb²⁺ disappears (Weng, 2004). As shown in Figure 1(a), the RR and removal capacity of Pb(II) depended greatly on pH. The low pH caused a large number of hydrogen ions in the solution to compete with Pb(II) for the negatively charged sites on the bacteria and fermentation products in the FBA9, and the H⁺ ions outcompeted the other ions. Hence, a low RR and removal capacity of Pb(II) were observed. The decreasing levels of H⁺ ions in solution as pH increased gave rise to Pb(II) being the dominant cationic species in solution; therefore, the RR and removal capacity of Pb(II) increased with pH. As the pH increased, the repulsive force between a large number of OH⁻ in the solution and the negative group in the fermentation liquid caused the metabolites in the fermentation liquid to extend fully and provide more active sites for Pb(II) capture. In addition, Pb(II) precipitates in the alkaline environment contribute to Pb(II) removal. Considering the precipitation tendency of lead solution when pH reaches 6, the best Pb(II) RR and removal capacity reached 93.1% and 310.3 mg/g, respectively, and the best pH was 5.0.

As shown in Figure 1(b), the RR of Pb(II) increased above 90% when 1% (v/v) FBA9 was added, then slowly increased after a slight decrease as FBA9 dosage increased; however, the removal capacity of Pb(II) decreased gradually after a slight increase at the same time. A lower dose of FBA9 meant less metabolic active substances removed Pb(II), and most of them were replaced by large levels of Pb(II). Further increases in FBA9 led to a gradual increase in the RR of Pb(II) because more active substances would provide more binding sites, which is advantageous to Pb(II) removal. Moreover, small particles were dispersed and could not be removed while large dosages of FBA9 wrapped on the surface of particles, causing a decreased removal capacity of Pb(II), with a maximum value of 1510 mg/g achieved at an FBA9 dosage of 1%. Thus, a value of 5% was selected as the best dosage of FBA9 based on the removal efficiency.

As shown in Figure 1(c), the RR and removal capacity of Pb(II) increased as the contact time increased from 5 to 45 min, the maximum RR and removal capacity of 94.2% and 314 mg/g were attained when the contact time was 35 min, and there was almost no significant increase in Pb(II) removal from 10 to 45 min. Most of the Pb(II) ions present in the solution interacted with the negatively charged sites of FBA9, facilitating the high RR. Progressive occupation of these negatively charged sites after 10 min resulted in total available binding sites being limited; hence, the removal process attained equilibrium. Thus, 10 min was selected as the best contact time for Pb(II) adsorption from the solution by the FBA9, which is much lower than the results reported in earlier previous studies of the adsorption of metal ions on various biomasses or bioflocculants (Feng et al., 2013).

As shown in Figure 1(d), the RR and removal capacity of Pb(II) under different temperatures were relatively small, with the highest values of 95.1% and 317 mg/g, respectively, being reached at a temperature of 30°C. These findings indicated that the process of FBA9 capturing Pb(II) had a wide range of temperatures, and 25–35°C was the best temperature.

Flocculation mechanism

FESEM analysis

To further investigate the removal process, the original and Pb(II)-loaded FBA9 were observed under the FESEM. Micrographs showed more complex structures and indicated that the material composition of Pb(II)-loaded FBA9 consisted of rod-shaped microorganisms, needle form products, and petal form products (Figure 2(a)). The energy spectrum analysis of the typical parts shown in Figure 2(a), after capturing Pb(II) ions, is shown in Figure 2(b) and (c).

Figure 2.

(a) FESEM analysis of Pb(II) removed by FBA9, (b) morphology and energy spectrum analysis of bacterial somatic cells, (c) energy spectrum analysis of needle-like morphological products, and (d) morphology and energy spectrum analysis of MBFA9 before and after capturing Pb(II) ions.

The morphology of the target organism of this experiment, Paenibacillus sp. A9, before and after capturing Pb(II) is shown in Figure 2(b). Scanning electron microscopy revealed the surface of the bacterial cells was smooth and flat before the removal of Pb(II) but that bright spots were present after the removal of Pb(II), and the surface of bacteria cells became rough and uneven as a result of adsorption of Pb(II) onto the surface of the bacteria cells. However, its adsorption capacity was limited, and morphological changes were only observed on the surface of a small number of bacteria cells. The energy spectrum peaks of Pb(II) are also shown in Figure 2(b). Some Pb elements, accounting for 8.23% of total elements, existed on the surface of bacteria cells indicating that the cells may contribute to the removal of Pb(II) by means of adsorption.

The process and mechanism of bacterial adsorption and detoxification of heavy metals are very complex, and they often occur through a variety of mechanisms (Wang et al., 2016). During the capture of Pb(II) by FBA9, the variety of structural components present in FBA9 means that many functional groups (e.g. carboxyl, amino, hydroxyl groups included in polysaccharides, protein, and small molecule acids) are able to interact with metal species. It is likely that the various mechanisms involved in Pb(II) removal, including cell surface adsorption, organic acid coordination reaction, and biological flocculation processes, occur simultaneously.

Heavy metal ions can react biochemically with some substances on the surface of bacteria. The outermost components of the cell wall, such as peptidoglycan, proteins, and lipids, can provide chemical carboxyl, hydroxyl, carbonyl, amino, mercapto, amide, phosphoryl, sulfate, and imidazole groups that form metal complexes that are adsorbed and fixed onto the cell surface (Yang et al., 2012). At the same time, some ions contained on the cell surface can exchange with heavy metal ions, thereby adsorbing and fixing heavy metal ions (Ye et al., 2013). For example, during the adsorption of Cd(II) by B. cereus, K⁺, Ca²⁺, Na⁺, and Mg²⁺ were found to be released, indicating that ion exchange promoted the process (Li et al., 2010). Ion exchange is one of the main mechanisms of bacterial surface adsorption; therefore, in this study, the bacterial cells may have contributed to the RR by complexation and ion exchange adsorption to fix heavy metal lead ions on the bacterial surface.

After the removal experiment, a large number of needle-shaped products were observed upon scanning electron microscopy (Figure 2(c)). There were obvious energy spectrum peaks of Pb in the energy spectrum figure of the needle products, and the quality of Pb elements accounted for 16.5%, indicating that it had a good effect on capturing Pb(II). It was speculated that these needle products that contributed to the removal of Pb(II) were the reaction products of Pb(II) and small molecules of organic acid alcohol produced by bacterial metabolic processes. Therefore, the acid alcohol metabolites contained in the fermentation fluid contributed to the removal of Pb(II).

During fermentation and culture, bacteria can secrete some metabolites to the extracellular environment. Small molecular organic acids such as malic acid, succinic acid, and citric acid are detected in FBA9. These organic acids and polymers have carboxyl, hydroxyl, and sulfonic groups, which easily form complexes with heavy metal ions (Mao et al., 2013; Qiu and Mao, 2013). Many studies of the removal of heavy metals by small molecular organic acids have found that different acids may promote or inhibit the removal of different heavy metals. Xia et al.(2018) showed that the RR of heavy metals by organic acids such as fumaric acid was affected by concentration, pH, and time. Lamelas et al. (2009) studied the effects of humic acid on the adsorption of Cd²⁺, Cu²⁺, and Pb²⁺ by algae cells and found that organic acid could promote the adsorption of Pb²⁺ because the complex of Pb–organic acid formed by Pb and small molecule organic acid in dissolved organic matter increased the hydrophobicity of Pb, after which it occupied a large number of active adsorption sites on the surface of algal cells and formed Pb–acid–algae. Ternary complexes enhance the penetration of Pb(II) into the cell membrane; therefore, because of the presence of malic acid, succinic acid, citric acid, and other organic acids in the FBA9, the acicular product formed after Pb(II) removal may be a binary complex of lead–organic acid that binds to adsorption sites on the cell surface to form a ternary system and coprecipitates under the flocculation of MBFA9.

At the same time, a large number of petal morphological products are visible in Figure 2(d), which were speculated to be the polysaccharide flocculation product MBFA9 produced during the metabolic process of the bacterial species. The results of previous studies showed that MBFA9 could have a good effect on flocculation and precipitation in various types of wastewater and that it was a highly efficient MBF. The morphology and energy spectrum analysis of the purified products of MBFA9 before and after capturing Pb(II) is shown in Figure 2(d). Scanning electron microscopy with a magnification of 5000× revealed that MBFA9 had a separate blocky structure with a rough surface before the removal of Pb(II). After capturing Pb(II), the structure became loose and expanded, and materials connected with each other internally and formed a large number of petal morphological products, which were denoted MBFA9–Pb. The energy spectrum also showed an obvious Pb(II) peak with an atomic percentage as high as 22.42%. Therefore, MBFA9, a polysaccharide MBF, plays an important role in capturing Pb(II) in FBA9.

Li (2017) reported that the adsorption isotherms of the capture of Pb(II) by FBA9 fit both the Freundlich and Langmuir equations well. A better fit with the Langmuir equation is an indication that the process of FBA9 capture of Pb(II) uses simple adsorption of a single molecular layer. FBA9 showed greater affinity for Pb(II), with a monolayer adsorption capacity of 189.9 mg/g of Pb(II) (Q, mg/g).

MBFA9, which consists of a large number of extracellular polysaccharides including mannose, glucose, acidic polysaccharides, and acetamino polysaccharides, was found in FBA9 (Jiang et al., 2014). MBFA9 participates via flocculation, mainly through hydroxyl, carboxyl, and amine groups that induce very high binding capacity through complexation and chelation. MBFA9 had a linear long chain molecular structure and appeared to have a molecular weight of 2.594 × 10⁶ Da. In this flocculation system, Pb(II) ions were electrostatically attracted and neutralized the anionic charges on the deprotonated carboxyl groups (–COOH) and hydroxyl groups (–OH) of MBFA9. MBFA9 was found to attach to several Pb(II) ions because of its polymeric nature, bridging them.

Overall, the results of scanning electron microscopy and energy spectral analysis of the fermented liquid before and after the removal of Pb(II) revealed that the mechanism of lead removal in the fermentation fluid occurred via three pathways, namely an adsorption effect of bacterial cells, combination with the small molecule acid alcohol metabolism product, and a flocculation effect of MBFA9.

FTIR spectral analysis

The results also indicated that MBFA9 can be successfully used for the removal of Pb(II) from aqueous solution. The FTIR spectra of the original and Pb-loaded MBFA9 in the range of 4000–400 cm⁻¹ are compared in Figure 3.

Figure 3.

FTIR spectroscopy of MBFA9 before and after capturing Pb(II) ions. a—MBFA9 before capturing Pb(II) ions and b—MBFA9 after capturing Pb(II) ions.

The spectrum of the Pb-free sample contained many absorption peaks, indicating the complex nature of the biomass examined. The absorption peaks in the range of 3600–3200, 3000–2850, 1660–1760, and 1250–1000 cm⁻¹ corresponded to stretching vibrations of O–H/N–H, C–H, C = O, and C–O groups, respectively. Moreover, the absorption peaks at 1357 cm⁻¹ corresponded to asymmetric and symmetric bending vibrations of C–H. In summary, the FTIR spectrum of the Pb-free MBFA9 showed the presence of hydroxyl, carboxyl, and amine groups, which played important roles in the removal of Pb(II).

After Pb(II) capture, many absorption peaks were obviously shifted with changes in wave number and intensity, suggesting the aforementioned functional groups were primarily involved in the adsorption of Pb(II) onto MBFA9. The shift from 3435 to 3422, 1667 to 1642, 1356 to 1403, and 1000 to 979 cm⁻¹ suggested that chemical interactions between the Pb(II) and O–H/N–H, C = O, C–H, and C–O groups occurred on the surface of MBFA9. Additionally, the shifted peak intensity mentioned above suggested that the reduction of the electron density of the functional groups further changed their vibration frequency and intensity. Moreover, the changes in peaks in the range of 800–400 cm⁻¹ might have been a result of ion exchange of carboxylic groups. In conclusion, the aforementioned variations in the absorption peaks suggest that hydroxyl, carboxyl, and amino groups and MBFA9 played a key role in the capture of Pb(II).

Conclusion

Using food processing wastewater fermented by flocculant-producing bacteria to partly replace carbon and nitrogen sources of fermentation medium and microbial FBA9 in the process of capturing heavy metal Pb(II) can enable the reuse of wastewater while greatly reducing the cost of microbial culture. Furthermore, the FBA9 appears to be an effective and alternative agent for the removal of Pb(II), and the maximum RR of 95.1% and removal capacity of 317 mg/g could be reached when FBA9 was added at a dosage of 5%, temperature of 30°C, pH of 5, and contact time of 35 min.

Use of FBA9 prepared from food processing wastewater reduced the cost and increased the yield of flocculant while also employing the combined action of bacterial cells, small molecular organic acids, and MBFA9 in FBA9 to achieve the effective removal of heavy metal Pb(II) ions from wastewater. Accordingly, the method presented herein will lead to the development of new processes for the treatment of heavy metal wastewater and should be explored at commercial and industrial scales.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This study was funded by the National Key Research and Development Plan Subject for Monitoring, Early Warning and Prevention of Major Natural Disasters (2017YFC1503105), and the National Natural Science Foundation of China (No. 51278090).

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