3.1 Isolation of high flocculating activity strains from ARTP mutation
A high cell lethality rate is desirable for efficient generation and selection of mutants. The effect of different ARTP treatment durations ranging from 10 to 100 s on the mortality rate of P. fluorescens is shown in Fig. 1A. According to previous reports, 90% of cell lethality percentage was set as the standard for the mutant generation (Hua et al., 2010; Liu et al., 2015). Therefore, 60 s was chosen as the appropriate exposure time in the following experiments. After plasma radiation treatment, the strain was propagated and cultured on a plate for 48 h. The mutant library was constructed from approximately 800 bacterial mutants. Developing an efficient pre-screening process to screen for desirable mutant strains is extremely crucial. The colony appearance of high-production EPS mutants showed the characteristics of “ropy” strand (Madiedo and Gavilan, 2005), which can be used to screen mutants. Colonies of a total of 106 mutants of strain T4, named T4-1 to T4-106, were picked out from the mutant library using “ropy” strand, then transferred to 250-mL Erlenmeyer flasks containing 50 mL fermentation medium and placed on a rotary shaker (150 rpm) at 30°C, incubated for 48 h to determine the flocculating activity. Compared with the original strain, 4 mutants (T4-2, T4-18, T4-20 and T4-75) exhibited considerably greater flocculating activity (Fig. 1B). The highest flocculating activity (568.49 U/mL) of mutant T4-2 achieved an increase of 106.48% than the initial strain. In addition, mutant strain T4-2 was grown continuously to investigate its genetic stability. The mutant strain T4-2 exhibited the same growth and flocculating activity after several operations up to 10 rounds (Fig. 1C), reflecting that the mutant strain has favorable genetic stability in EPS production.
3.2 Optimization of medium components and culture conditions of mutant T4-2
The secretion of microbial metabolites during fermentation depends not only on the genetic characteristics of the microorganism, but also on the composition of the medium. As previously described, there is an appreciable impact on bacterial growth and EPS yield when choosing different carbon and nitrogen sources for fermentation (Li et al., 2009; Salehizadeh and Yan, 2014). It is well known that metal cations are one of the most vital factors influencing the production of EPS as it plays a crucial role in enzymatic reactions involved in EPS synthesis (Salehizadeh and Yan, 2014).The results for the various carbon, nitrogen sources, and metal cations affecting the flocculating activity were described in Table 1. One noteworthy result was that mutant T4-2 grew well and produced EPS with all the carbon sources assayed. More importantly, glucose is the most efficient source of carbon for bacterial growth of mutant T4-2 and EPS production. Yeast extract and urea were chosen to be the optimal complex nitrogen source for EPS production as they created the highest flocculating activity (593.7 U/mL), which is significantly higher than those with alternative nitrogen sources. As shown in Table 1, Mn2+ is chosen as the optimal cation for EPS production by mutant T4-2. Due to the great influence on microbial metabolism, especially the production of EPS, the C/N ratio associated with EPS production has received great attention (More et al., 2014). From this, the concentrations of glucose, urea, and yeast extract were determined, and the results were shown in Figure S1. In addition, the effect of the concentration of inorganic salts and metal cations on EPS production was investigated and the results were shown in Figure S2. Based on the experimental results, the optimal medium component was obtained as follows (g/L): glucose 15.0, yeast extract 0.8, urea1.0, KH2PO4 0.1, K2HPO4 0.1, MnCl2 0.1, NaCl 2.0.
Culture temperature and initial media pH, inoculum size are also important factors influencing flocculating activity and EPS production. The optimized culture conditions were shown in Figure S3. The flocculating activity reached 2579.9 U/mL under these conditions, and the purified EPS yield reached 4.84 g/L. Besides, it was found that the cell-free supernatant possessed nearly 95% flocculating activity (2450 U/mL), while cells only displayed flocculation activity of 28 U/mL. Following the previous findings (Subudhi et al., 2016; Xia et al., 2008), the cell-free supernatant was found to have greatly higher activity than the culture pellet, indicating that the EPS produced by P. fluorescens mutant T4-2 has flocculating activity.
Table 1
Effect of different carbon sources, nitrogen sources and cations on the production of the EPS from P. fluorescens mutant T4-2
Carbon source | FA ± SD (U/mL) | OD600 | Nitrogen source | FA ± SD (U/mL) | OD600 | Cation | FA ± SD (U/mL) | OD600 |
---|
Sucrose | 519.5 ± 21.1 | 4.00 ± 0.01 | Urea | 36.1 ± 13.3 | 0.68 ± 0.04 | Mg2+ | 1264.1 ± 26.4 | 3.56 ± 0.13 |
Fructose | 243.5 ± 9.9 | 4.69 ± 0.02 | NaNO3 | 26.5 ± 5.1 | 0.50 ± 0.04 | Ca2+ | 1257.5 ± 15.1 | 4.20 ± 0.12 |
Glucose | 583.7 ± 5.1 | 5.23 ± 0.06 | NH4Cl | 179.8 ± 35.2 | 3.73 ± 0.02 | Mn2+ | 1285.1 ± 12.4 | 4.22 ± 0.09 |
Glycerol | 26.1 ± 6.6 | 3.12 ± 0.12 | Yeast extract | 31.4 ± 4.4 | 1.84 ± 0.21 | K+ | 623.4 ± 19.1 | 4.26 ± 0.02 |
Lactose | 274.4 ± 7.3 | 4.38 ± 0.15 | Tryptone | 151.3 ± 21.7 | 3.00 ± 0.09 | Fe3+ | 0 ± 0 | 3.32 ± 0.13 |
Maltose | 259.3 ± 19.6 | 4.62 ± 0.04 | Beef extract | 201.5 ± 32.1 | 2.26 ± 0.15 | Al3+ | 618.4 ± 20.0 | 4.33 ± 0.21 |
Xylose | 256.5 ± 17.4 | 4.53 ± 0.04 | Soy flour | 56.7 ± 7.4 | 1.09 ± 0.14 | Zn2+ | 32.5 ± 3.1 | 2.97 ± 0.24 |
Sodium Citrate | 229.5 ± 23.0 | 4.83 ± 0.04 | Mixed Nitrogen | 593.7 ± 5.1 | 4.91 ± 0.09 | Cu2+ | 2.7 ± 1.8 | 0.18 ± 0.02 |
* FA = Flocculating activity, SD = Standard deviation, mixed nitrogen = [yeast extract + urea] |
3.3 Fermentations of mutant T4-2 on 3.6-L bioreactor
To evaluate the performance of the P. fluorescens mutant T4-2 under a more stable conditions, batch fermentation was performed in a 3.6-L bioreactor with optimal culture media and culture conditions. As shown in Fig. 2, the flocculation activity and biomass were significantly improved. During the exponential phase, dissolved oxygen decreased rapidly to about 10%, and then continued to decline to 0 at 6 h, indicating that the growth of bacteria needs a lot of oxygen. After 28 h of fermentation, the glucose in the medium was practically exhausted and the biomass reached a maximum value of 7.96 g/L at 32 h and a maximum flocculation activity of 3023.4 U/mL at 46 h, suggesting that EPS is a secondary metabolite. During the later stages of fermentation, the flocculating activity and biomass decreased, which possibly some of the bacteria were autolyzed when nutrients were used up, then various enzymes in the cell were exposed. The hydrolysis of the EPS by these enzymes results in the decrease of flocculation activity (Yu et al., 2016). The flocculating activity of mutant T4-2 in 3.6-L bioreactor reached 3023.4 U/mL, which was 17.19% higher than that in conical flasks (2579.9 U/mL). The final yield of EPS reached 6.42 g/L, 34.1% higher than that of the cultivation in conical flasks (4.84 g/L). The result is greatly higher than most EPS-producing strains, such as Aspergillus flavus (Aljuboori et al., 2013), Bacillus sp. (Okaiyeto et al., 2015) and Proteus mirabilis TJ-1 (Xia et al., 2008).
3.4 Characterization of EPS
The purified EPS was mainly composed of polysaccharide (76.27%) and protein (15.8%). Further analysis showed a mass ratio of 13:7:1 with the neutral sugar (34.43%), uronic acid (19.84%) and amino sugar (2.71%). It was observed that uronic acid and amino sugar make most of the contribution to the flocculation capacity of the EPS molecule, attributing to their carboxyl groups and amide groups, which are conducive to adsorption particles and flocculation (Gao et al., 2006). Tiwari et al. also reported that there was a positive correlation between uronic acid content and flocculation activity (Tiwari et al., 2015).
The functional groups of the purified EPS were identified using FT-IR spectroscopy. The result of FT-IR was shown in Fig. 3C and consistent with the previous study (Xiong et al., 2010), indicating the presence of hydroxyl, amide and carboxyl groups in the EPS. Elemental analysis showed that EPS from P. fluorescens mutant T4-2 contained C, H, N, 29.64%, 5.24% and 5.20% respectively. The average molecular weight of the EPS was 1.17×105 Da, which is significantly higher than other EPS reported in the literature (Aljuboori et al., 2013; Li et al., 2010; Li et al., 2009). High molecular weight contributed to the flocculating activity because of extra adsorption points and stronger bridging (Giri et al., 2015; Kumar et al., 2004).
3.5 Performance of the EPS in chromium(Ⅵ) removal
3.5.1 Effect of the solution pH
The initial pH of the solution has a major impact on the adsorption process of heavy metal ions. It affects not only the charged surface functional group of EPS, but also the form of chromium in solution. As shown in Fig. 4A, the effect of pH on chromium(Ⅵ) adsorption by the EPS and zeta potential of the EPS were investigated. The zeta potential of EPS decreases from − 2.3 to -30.2 as the pH increases from 2 to 9, indicating that the surface charge of EPS is negatively charged. The species distribution of chromium (Ⅵ) was shown in Fig. 4B, chromium (Ⅵ) exists by two anions of HCrO4− and Cr2O72− in the range of pH 2–9. From Fig. 4A, it shows that the adsorption capacity of the chromium(Ⅵ) was found to decrease with the increase of the initial pH of the contact solution. However, large amounts of protons have a positive effect for promoting the reduction of Cr(VI) to Cr(III) at low pH. Cr(III) mainly exists in acid solutions in the form of three cation of Cr3+, CrOH2+ and Cr(OH)2+. The negatively charged EPS surface was easy to bind with the positively charged ionic groups of Cr(III) through electrostatic attraction, thus reducing the Cr(VI) anionic species in the solution. On the other hand, it is reported that amides (–NH–) on the extracellular polymer reduced part of Cr(VI) (82.3%) to Cr(III) (Wei et al., 2015). We supposed that part of the Cr(VI) is reduced to Cr(III) in this study because EPS contains amide (-NH-) groups from FT-IR spectroscopy result (Fig. 3C). Generated Cr(III) immobilize on the surface of the EPS by electric neutralization, thereby reducing the content of Cr(VI) in the solution and improving the adsorption efficiency. The adsorption of chromium (Ⅵ) by EPS decreased from 80.13–30.67% as the pH increased from 2 to 9. An acidic environment is preferred for effective chromium (Ⅵ) adsorption (Guo and Chen, 2017), the highest adsorption capacity of chromium (Ⅵ) reached as high as 80.13 mg/g at pH 2. These results (Fig. 4) demonstrated that the EPS exhibited a high adsorption affinity toward chromium (Ⅵ).
3.5.2 Adsorption isotherm and kinetics for chromium (Ⅵ) removal
Adsorption isotherm can reflect the distribution of adsorbed molecules in the liquid phase and solid phase in equilibrium at a certain temperature; it is an essential index to evaluate the adsorption performance of adsorbent. Langmuir, Freundlich and Redlich Peterson adsorption models were studied to simulate the adsorption of hexavalent chromium and by the extracellular polymeric substance (EPS). According to the fitting results of three models to the experimental data as Fig. 5 (A-C) shows, the parameters (qm, B, KF, n, K, α, β) and correlation coefficient R2 are calculated, the calculated results are given in Table 2. The Langmuir model has the highest fitting correlation coefficient for chromium (Ⅵ) adsorption process, which implies that the adsorption of hexavalent chromium by the EPS belongs to homogeneous adsorption, and the adsorption sites do not interfere with each other. Once the adsorption is completed, it is no longer affected by the absorbent. The analysis of extracellular polymeric substance for removal chromium (Ⅵ) in aqueous shows that the above three adsorption isotherm models can better fit the adsorption process (R2 > 0.91). It shows that the adsorption process of EPS for chromium (Ⅵ) is complex and may involve several adsorption mechanisms. This may be due to peculiar structure of EPS, such as porous structures that may lead to multi-step adsorption processes. The parameters qm and B in the Langmuir adsorption isotherm model decrease with increasing temperature, indicating that the adsorption chromium (Ⅵ) of by the EPS is an exothermic process.
Table 2
Isotherm parameters for the adsorption of chromium (Ⅵ)
| T | Langmuir isotherm | Freundlich isotherm | Redlich-Peterson isotherm |
(K) | qm (mg g − 1) | B (L mg− 1) | R2 | KF (mg g − 1) | n | R2 | K | α | β | R2 |
Cr(VI) | 298 | 83.89 | 0.146 | 0.994 | 24.18 | 3.510 | 0.978 | 61.805 | 1.984 | 0.775 | 0.975 |
308 | 76.51 | 0.142 | 0.995 | 27.025 | 3.597 | 0.920 | 27.723 | 0.512 | 0.888 | 0.914 |
318 | 71.32 | 0.136 | 0.994 | 32.292 | 3.964 | 0.984 | 128.730 | 3.417 | 0.784 | 0.981 |
Adsorption kinetics is the relation between the adsorption capacity and the adsorption time, which is an essential feature to describe the adsorption efficiency. Kinetic models of pseudo-first-order, pseudo-second-order and were used to simulate the dynamical experimental data, and the experimental results were shown in Fig. 5 (D-F). For evaluating the type of reaction mechanism involved, according to the fitting results of the dynamical experimental data, the parameters (k1, qe, k2) and correlation coefficient R2 of each model were calculated, and the calculated results were shown in Table 3. For the adsorption of hexavalent chromium by extracellular polymer substance, the pseudo-second-order kinetic model was a better fit than the pseudo-first-order kinetic model. The theoretical equilibrium adsorption capacity qe of hexavalent chromium is 90.90 mg/g from Table 3, which is closer to the experimentally measured equilibrium adsorption capacity of 80.13 mg/g. The results also indicate that the rate-limiting step of the adsorption process maybe chemical adsorption, which is consistent with the results of previous studies on the removal of heavy metal ions by flocculant (Fan et al., 2019). FTIR analysis indicated the presence of hydroxyl, amide and carboxyl functional groups in the EPS, which possibly provide active sites in the aggregations of hexavalent chromium (Xu et al., 2012).
Table 3
Kinetic parameters for the adsorption of chromium (Ⅵ)
T(K) | Pseudo-first-order kinetic | Pseudo-second-order kinetic |
---|
k1(min–1) | qe(mg g–1) | R2 | k2(mg g–1·h–1) | qe(mg g–1) | R2 |
---|
298 | 0.055 | 78.70 | 0.867 | 0.743×10− 3 | 107.52 | 0.989 |
308 | 0.058 | 66.83 | 0.849 | 0.853×10− 3 | 91.58 | 0.996 |
318 | 0.050 | 53.08 | 0.926 | 0.942×10− 3 | 77.28 | 0.995 |
3.5.3 Thermodynamic Parameters and Activation Energy
The thermodynamic parameters, namely, the Gibbs free energy change (ΔG0), standard enthalpy change (ΔH0), and standard entropy change (ΔS0), were calculated according to the Van't Hoff equation (Table S1) to evaluate the adsorption thermodynamic behavior. The values of the thermodynamic parameters for Cr(VI) adsorption onto EPS are given in Table 4. The Gibbs free energy change ΔG0 were negative at various temperatures, confirming that the adsorption was spontaneous and thermodynamically favorable. The absolute value of ΔG0 gradually increased with the increase of temperature, which proved that the increase of temperature was beneficial to the adsorption process. The negative values ofΔH0 indicated that adsorption was exothermic, which agrees with the results of Langmuir adsorption isotherm model. The positive values of the entropy ΔS0 suggest that increasing randomness during the adsorption process (Xu et al., 2012). Activation energy is the amount of energy required for a molecule to transition from a normal state to an active state where chemical reactions can easily occur. The value of activation energy Ea was calculated is 9.36 kJ mol− 1 from the Arrhenius equation (Table S1). It affirmed that the physisorption phenomenon is the prevailing react because the activation energy is in the range of 5–40 kJ mol− 1. (Wei et al., 2015)
Table 4
Activation Energy and Thermodynamic Parameters for Adsorption of chromium (Ⅵ)
Ea(kJ mol–1) | ΔG0(kJ mol–1) | ΔH0(kJ mol–1) | ΔS0 (J mol–1K–1) |
---|
298K | 308K | 318K |
---|
9.36 | -21.83 | -22.21 | -22.49 | -2.787 | 82.75 |
Recently, EPS has attracted more researchers' interest because of their heavy metal removal properties (Li et al., 2022; Siddharth et al., 2021). Due to different chemical structures and functional groups, EPS from different strains have different performance in removing heavy metals. The reported adsorption of heavy metals by EPS mainly focused on the adsorption of Cd(II) (Yin et al., 2013), Cu(II) (Wang et al., 2014), Pb(II) (Kumari et al., 2017), Zn(II) (Li et al., 2019)and Ni(II) (Nkoh et al., 2019), but there are few reports on the adsorption of Cr(VI). The EPS from Pseudomonas fluorescens in this study is better than most reported in adsorption of Cr(VI) (Paul et al., 2012; Pi et al., 2021). The adsorption process is a popular, commonly adopted and efficient process for the removal of Cr(VI) from aqueous solutions. As an environmentally friendly and easily degradable Cr(VI) adsorbent, EPS in this study is an adsorbent with potential industrial application whether used directly or with modification. However, this EPS for other heavy metals removal is still under investigation.