Immobilized Microbial Catalytic Oxidation Preparation and Application of Biopolymeric Ferric Sulfate

A novel inorganic polymer flocculant, biopolymeric ferric sulfate (BPFS), was prepared by immobilization technology of microorganisms and by oxidation of ferrous sulfate using domestic iobacillus ferrooxidans (T. f ) under acidic condition. T. f was isolated on the agarose single-plate medium, which exhibited an unusual trait on the utilization of low concentration of the nitrogen source and phosphorus as the nutrient substance. Under the optimal conditions, the microorganism could grow and reproduce normally and maintain the strong catalytic oxidation activity to Fe.)e immobilization of T. f on the polyurethane as the support matrix was investigated. Cycling batch operation was applied to the preparation of 40 kg/m, 60 kg/m, and 80 kg/m BPFS when the optimal conditions are pH value of 1.8, circulation flow rate of 0.28–0.30 L/h, and reaction temperature of 28 ± 1°C. When the prepared BPFS and SPFS (solid biopolymeric ferric sulfate) were used to dispose Songhua River water, the removal rate of turbidity and CODMn of BPFS was slightly better than that of SPFS. )e removal efficiencies of turbidity and CODMn by BPFS could reach 93.9% and 79.7%, respectively. )e result suggests that the BPFS has good flocculating activity.

ese production methods are under harsh production conditions, long period, high cost, and oxidant residue resulting in serious corrosion on the dosing system [8].In order to overcome these shortcomings, biotechnology was introduced into the preparation of PFS.
e product called biopolymeric ferric sulfate (BPFS) was prepared by catalytic oxidation of microorganisms.BPFS has good turbidity and COD removal, its preparation conditions are mild, production costs are low, the process is no pollution, and microbial secretions can play a secondary flocculation [9].
iobacillus ferrooxidans (T.f ) is Gram-negative chemolithotrophic bacterium.T. f with CO 2 as the carbon source and nitrogen and phosphorus as nutrients can obtain energy from the oxidation of ferrous iron and sulfur [10,11].T. f has been widely utilized in the processes of bioleaching, the desulphurization of coal and environmental pollution control.In recent years, there are many researches on their leaching capacity and their own metabolic activities.Arsenic (As) leaching was conducted from highly concentrated As mine tailings using the ironoxidizing bacteria, and the pH range should be controlled to prevent the formation of jarosite [12].More recently, T. f has been widely utilized in the processes of preparation of BPFS at normal temperatures and pressures [13].At present, the methods of preparation of BPFS by biocatalytic oxidation have many advantages such as wide sources of raw materials and easy operation, but the easy loss of bacteria leads to the low utilization rate of bacteria and production efficiency and the long period.
In order to further improve the reaction rate and shorten the reaction cycle, the preparation of BPFS by the microbial immobilization technology was studied [14].In this study, BPFS was prepared by the microbial immobilization technology on the polyurethane as the support matrix.e in uence of aeration and nutrient factors on the oxidation rate of Fe 2+ was also investigated.e growth of T. f in low concentration of nutrient conditions was studied to optimize the preparation of BPFS.At the same time, its application in water treatment was also investigated.is study provides the theoretical basis for the further industrialization of BPFS and the expansion of application elds.
ese three solutions were autoclaved/sterilized separately and mixed when the temperature of the autoclaved solution was cooled to 45 ± 1 °C [15].

Enrichment and Isolation.
T. f was enriched in the solid medium Z successively and incubated in a 30 ± 1 °C incubator.e pure isolate was obtained after many times of sequential respreadings [16].

e Molecular Biology Identi cation of iobacillus ferrooxidans.
e genomic DNA was extracted from the bacterial genome, ampli ed by PCR, and ampli ed. e recovered fragment was sent to Ming Chen Zhiyuan Biotech Company for sequencing.e sequencing primers were 16S PCR primers.Sequence identi cation was performed at the BLAST facility of the National Center for Biotechnology Information (NCBI) to identify microorganisms.

2.4.
Culture of the Strain.T. f was enriched in the 9K medium at 30 ± 1 °C under shaking condition (150 rpm).Short rodshaped bacterium was observed into the 9K liquid medium after logarithmic growth.
e in uence of pH and concentration of nitrogen and phosphorus in solution on catalytic oxidation of Fe 2+ activity was also investigated.

Preparation of BPFS.
In this experiment, a bioreactor with reticulated polyurethane as the carrier was used to carry out the immobilization experiment of the microbe and the preparation experiment of the polyferric sulfate by repeated batch operation [17,18].e preparation apparatus is shown in Figure 1.
Based on breeding selection and domestication, T. f was selected as the biocatalyst to prepare BPFS.Cycling batch operation results in the preparation of 40 kg/m 3 , 60 kg/m 3 , and 80 kg/m 3 BPFS when the optimal conditions are circulation ow rate 0.28-0.30L/h and reaction temperature 28 ± 1 °C.

Flocculation Experiments.
To evaluate the occulation e ect of 40 kg/m 3 , 60 kg/m 3 , and 80 kg/m 3 BPFS and SPFS, occulation experiments tested winter Songhua river (T 1 ± 1 °C, pH 7.52, turbidity 2.61 NTU, COD Mn 6.21 mg/L) in a jar test apparatus.Afterwards, the suspension was agitated at speed of 150 r/min for 60 s and at speed of 70 r/min for 20 min; then, it was left undisturbed for 25 min, and the supernatant sample was collected for further analysis.

Isolation and Molecular Biology Identi cation of the
Strain.Round red-brown colonies on the culture were observed on the surface of the solid medium Z after two weeks.And then after single-layer coating on the separation, a large colony was selected and repeatedly puri ed in the 9K liquid medium.e agarose single-layer plate method was used for 16S rDNA sequence analysis and identi cation, the sequence length of 1469 bp (Figure 2).
iobacillus ferrooxidans is strictly autotrophic organisms, and most of the chemoautotrophic organisms are highly sensitive to organic compounds, such as polysaccharides and other components in agar.
e small molecules are produced by the agar under acidic condition.However, saccharomycetes can metabolize the small molecules produced by the agar and inhibit the growth of T. f on the solid medium.erefore, the modi ed solid medium used agarose instead of agar to gel solidify the medium, and this can support the growth of most strains [19,20].
e sequence homology was compared with that of NCBI by Blast.e results showed that the strain had high homology with Acidithiobacillus sp.E6-1, 3-8 HM769771.1 (>99.5%), and it can be concluded that the strain belongs to the genus iobacillus acidophilus.e strain was fermented   3. e produced precipitates were dried and weighed, and the obtained precipitates were 0, 0.33, 1.92, 3.64, and 4.78 g/L, respectively.e preliminary analysis of the precipitate showed yellow potassium ferrite and some metabolites of microorganisms, during the process of microbial growth and reproduction.Due to the increasing Fe 3+ content, it easily reacts with M + (where M may be K + , NH 4+ , Na + , etc.), HSO 4
According to Figure 3, the decrease in biooxidation is related to the inhibition of bacterial activity by the pH value in a certain range.When pH ≤ 1.2, Fe 2+ content keeps steady, due to partial enzyme of T. f inactivation, making the thallus not to carry out the complete life activities, grow, and multiply.erefore, Fe 2+ cannot be oxidized to Fe 3+ , and the reaction cannot continue to occur.When 1.2 < pH < 1.6, it can inhibit the growth of bacteria, and the average oxidation rate of Fe 2+ decreases.When 1.6 ≤ pH ≤ 2.0, with the increase of catalytic activity of T. f, the average oxidation rate of Fe 2+ increases.As pH increases, more precipitation is generated.In addition, considering reagent usages and generating precipitation, the initial pH value of 1.8 is considered to be appropriate for the preparation of BPFS.

In uence of Nitrogen and Phosphorus Source
Concentration.
e e ects of two nutrient contents on microbial growth and the Fe 2+ oxidizing e ciency were investigated.e results are shown in Figure 4.As shown in Figure 4, under the catalysis of microbes, reddish-brown BPFS was synthesized through 9 g/L and 40 g/L of industrial ferrous sulfate solution as the energy source, both at low concentration of nutrients and normal concentration of nutrients.It is illustrated that, under low concentration of nutrients, the concentration of nutrients does not have much impact on the conversion of Fe 2+ and keeps good oxidative activity of bacteria.us, reducing the nitrogen and phosphorus concentration for the preparation of BPFS is chosen.

Adaptability of iobacillus ferrooxidans to Industrial Ferrous Sulfate.
e e ect of industrial ferrous sulfate as an energy source in the preparation of BPFS on microbial growth and the Fe 2+ oxidizing e ciency was investigated.
e results are shown in Figure 5.As shown in Figure 5, under the condition of low concentration of nutrients, with the increase of Fe 2+ concentration, the average oxidation rate of Fe 2+ decreases.Low Fe 2+ concentration cannot provide su cient energy to the growth of bacteria.However, too much Fe 2+ can inhibit the growth of bacteria, making microbial catalytic oxidation capacity to be reduced.Ferrous sulfate solution with high or low Fe 2+ content has a negative e ect on the activity of microbial catalytic oxidation, which is due to industrial sulfuric acid iron solution containing copper, zinc, manganese, and other impurities [21], inhibiting the growth of bacteria, making microbial catalytic oxidation capacity to be reduced.Based on the comparison of the growth of bacteria under di erent FeSO 4 conditions, considering reagent usages and production costs, it is concluded that industrial ferrous sulfate has a higher practical value.

Microbial Immobilization with Reticulated Polyurethane.
e adsorption and immobilization process of microorganisms in the reticulated polyurethane bioreactor was shown by the average oxidation rate of Fe 2+ in the previous 18 cycles.e results are shown in Figure 6.
As shown in Figure 6, the average oxidation rate of Fe 2+ increased with the reaction period in the immobilization (bio lm culturing) stage.When the average oxidation rate of Fe 2+ stabilized in two adjacent periods, the number of immobilized microorganisms was saturated.When the bio lm was formed completely, the average oxidation rate of Fe 2+ is up to 2.64 g/L•h and BPFS with good performance can be obtained.To optimize the preparation conditions of BPFS, experiments were conducted with di erent total iron concentrations of BPFS.

Preparation of BPFS
(1) e E ect of Aeration Rate.In order to improve the production e ciency, the e ect of aeration rate on the  Journal of Chemistry production e ciency was studied when the total iron concentration was 40 kg/m 3 , 60 kg/m 3 , and 80 kg/m 3 BPFS.e results are shown in Figure 7.
Figure 7 shows that the 40 kg/m 3 , 60 kg/m 3 , and 80 kg/ m 3 BPFS were obtained by the cycling batch operation, the optimum aeration rate were 1.40-1.50L/L•min, 1.80-1.90L/L•min, and 2.00-2.10L/L•min, respectively, and the average oxidation rates of Fe 2+ were 3.35 g/L•h, 2.51 g/L•h, and 1.91 g/L•h, respectively.With the increase of aeration amount, the average oxidation rate of Fe 2+ increases, and the production e ciency of BPFS is also improved.High concentrations of BPFS have a higher viscosity; in the low aeration, the oxygen cannot break through the liquid membrane, resulting in the microbial lack of adequate oxygen; growth and reproduction are limited, and the average oxidation rate of Fe 2+ decreased.In the high aeration, the reactor was lled with a certain amount of air so that a large number of microorganisms can grow and multiply, and the average oxidation rate of Fe 2+ was improved.

Journal of Chemistry
(2) E ect of Nutrient Substances.To investigate the e ect of the nutrient substances on the BPFS preparation, experiments were conducted with single-and double-nutrient substances.Single nutrient composition is as follows: 1 g/m 3 KCl, 40 g/m 3 (NH 4 ) 2 SO 4 , 20 g/m 3 K 2 HPO 4 , 20 g/m 3 MgSO 4 , and 1.5 g/m 3 Ca(NO 3 ) 2.
e content of double-nutrient substances is twice the single nutrients.
e results are shown in Figure 8.
According to Figure 8, with the increase of multiple nutrients, the catalytic oxidation capacity of microorganisms in the reactor increased, and the production e ciency of BPFS was improved.Under the conditions of single nutrients, microbial growth is limited because of insucient nutrient requirement for microbial growth and reproduction.us, the optimum nutrient content is doublenutrient substances.
(3) Reaction of Di erent Sampling Ports in the Bioreactor.Under optimal operating conditions, the Fe 2+ content in the four samples connection and outlet of the bioreactor was monitored simultaneously.e distribution of the results is shown in Figure 9.
Figure 9 shows that the Fe 2+ contents of the four sampling ports and the liquid outlet are close at the same When the polyurethane material is used as the carrier to immobilize microorganism, the average oxidation rate of Fe 2+ is higher than that of the best experimental results in the early stage of the study.At the early stage, in the preparation of BPFS of 40 kg/m 3 , 60 kg/m 3 , and 80 kg/m 3 by ceramic raschig ring as the carrier, the microbial concentration of the immobilized microorganism on the ceramic raschig ring was 1.35 * 10 8 cells/mL, 1.96 * 10 8 cells/ mL, and 1.11 * 10 8 cells/mL, respectively.e average oxidation rates of Fe 2+ were 3.35 g/L•h, 2.51 g/L•h, and 1.91 g/ L•h, respectively, and the concentration of the microbes with polyurethane immobilization was 2.31 * 10 8 cells/mL, 2.10 * 10 8 cells/mL, and 1.55 * 10 8 cells/mL.erefore, the use of polyurethane as a carrier to immobilize microorganisms can enhance catalytic oxidation of Fe 2+ and solve the problem of low utilization of bacteria to improve the production e ciency of BPFS.

Application of BPFS.
e occulating e ect of BPFS and SPFS is compared.
e results are shown in Figures 10  and 11.
Figures 10 and 11 demonstrate that the BPFS prepared in this study is superior with respect to the turbidity and COD Mn removal.When the dosage of BPFS was 13-20 g/m 3 , turbidity removal rate (above 90%) and signi cant COD Mn removal e ciency (above 70%) were found, as shown in Figure 10.
is is because BPFS not only has a high degree of polymerization but also contains microorganisms, which can catalyze the oxidation of organic matter as a condensation nucleus during the occulating deposition process.us, it is sticky and can improve the coagulation e ciency, resulting in the adsorption of big molecular organic matter [22].On the contrary, the gure shows that there is a sharp decrease in the treatment e ciency as the coagulant dosage increased to 20 g/m 3 .
e reason for the worsening performance with the increase of coagulant's concentration is the overdosing e ect, which is based on the chain bridging mechanism [23][24][25][26].With the addition of occulant, the surface zeta potential of the particles decreases, and the particles in the water begin to occulate.As the occulant dosage increases, there is a gradual increase in the zeta potential value, and it almost reaches the point of zero charge (0 mV) [27].Continuing to increase the amount of occulant dosage, the adsorption surface of the colloid is coated by occulant, which lost the opportunity to combine with other colloidal particles.So the surface zeta potential begins to rise, and the repulsion of colloidal particles increases [28][29][30].
erefore, achieving another relatively stable state, the emergence of the antistabilization phenomenon is hard to agglomerate, which results in deterioration of the occulation e ect [31][32][33][34].Less dose of occulant is not enough to form a bridge, and the excess dose of occulant deteriorates the function of the polymer occulant.erefore, in the water treatment process, adding appropriate amount of occulant improves its occulation e ect [35].

Conclusion
e immobilization of T. f on the polyurethane as the support matrix was investigated to improve the utilization  Journal of Chemistry rate of bacteria and increase the production e ciency of BPFS.
(1) Strain T. f was isolated on the agarose single-plate medium.Based on 16S rRNA sequence data, the bacterium was identi ed as T. f (2) A new preparation method of PFS was conducted using a T. f pH value of 1.8, circulation ow rate of 0.28-0.30L/h, double nutrient material, and reaction temperature of 28 ± 1 °C.Under the optimal conditions, the 40 kg/m 3 , 60 kg/m 3 , and 80 kg/m 3 BPFS product was obtained by cycling batch operation, the optimum aeration rate was 1.40-In this study, due to the limitation of time and conditions, with the increase of total iron content, the increase of raw liquor viscosity gradually becomes the main factor restricting the oxidation rate of Fe 2+ . is is a question that

Figure 9 :
Figure 9: Fe 2+ content in various parts of the bioreactor column.