Biofilm formation assay
Biofilm formation plays a vital role in metal corrosion. In this study, we assessed the biofilm forming capability of the promising anticorrosion B. cereus inoculum in polystyrene tubes. The biofilm formation of B. cereus inoculum after different incubation time is presented in Fig. 2a, the inset corresponds its digital photographs. As can be seen, the biofilm attached on polystyrene tube wall accumulate gradually, the OD598 increased from 0.02 to 0.81 during the 48h incubation, demonstrating the B. cereus inoculum exhibited a strong biofilm forming capacity. The inset view displayed a gradually deepening staining which illustrated the biofilm increases (Xu et al., 2020). Apparently, the biofilm induced violet color gradually became deeper with time.
Biocorrosion studies
Weight loss tests
Figure 2b, c and d shows the Q235 CS corrosion inhibition performances of B. cereus on 3 d rotation tests determined by weight loss measurement. Overall, the corrosion inhibition performances of the activated B. cereus inoculum supernatant were superior than B. cereus broth, especially at high concentration levels. Figure 2b comprehensively investigated the Q235 CS corrosion inhibition performances of B. cereus broth with respect to different growth periods. In general, the anticorrosion capacities of B. cereus broth were common, the corrosion inhibition efficiencies of log, stable, and decline B. cereus broth all decreased gradually with the increment of inoculation dosage. In the inoculum range of 0.025-1%, the corrosion inhibition efficiency of log phase B. cereus broth decreased from 54.32–32.31%, this efficiency range reduced to 54.95–16.32% for stationary phase broth and 52.19–23.73% for decline phase broth. The corrosion inhibition efficiencies of sterilized stationary phase B. cereus broth were stable, maintained about 50% in the inoculum range. In addition, contribution of LB medium was also considered, the inhibition performances of LB medium increased slightly with the increase of inoculum, and the highest inhibition rate was 54.58% at 1% inoculum. The corrosion inhibition efficiencies of LB medium were higher than B. cereus broth, indicated inoculated B. cereus did not enhance the Q234 CS anticorrosion properties.
Figure 2c shows the corrosion inhibition performances of B. cereus inoculum in another activation approach. As it can be seen, the B. cereus inoculum had excellent corrosion inhibition capacities on Q235 CS, regardless of 1h or with substrate 8h activation, their corrosion inhibition efficiencies increased continuously. In tested concentration range of 50-2000 mg/L, the anticorrosion efficiencies increased from 40.94–88.59% for 1h activation and 63.09–92.84% for with substrate 8 h activation, respectively. In contrast, the B. cereus inoculum after sterilization showed a first decreasing and then a weakly increasing anticorrosion performance. The anticorrosion efficiencies of sterilized 1h activated B. cereus inoculum were 57.69% at 50 mg/L, decreased to 39.09% at 1000 mg/L and then slightly increased to 52.16% at 2000 mg/L, the corresponded anticorrosion efficiencies of sterilized 8 h activated B. cereus inoculum was 68.53% at 50 mg/L, slightly reduced to 60.38% at 1000 mg/L and later rose to 83.54% at 2000 mg/L. The substrate (brown sugar + urea) used for the 8 h activation was also examined and its corrosion inhibition efficiencies maintained about 55% (+-5%). The above results demonstrated at higher concentrations, the survival of B. cereus contributes to Q235 CS corrosion protection.
The 3 d rotation tests were conducted for primary activation screening. According to the results, 8 h activation with substrate exhibited the optimal corrosion inhibition performance and therefore was selected for subsequent 14 d rotation tests (Fig. 2c). The compared 3 d and 14 d corrosion performances of the B. cereus inoculum after activating 8h with substrate are showed in Fig. 2d. The results showed that higher the concentration of B. cereus inoculum added, the higher the corrosion inhibition efficiencies. The anticorrosion effects were similar between 3 d and 14 d rotation tests at lower concentrations (50–600 mg/L) but obvious distinct at higher concentration (2000 mg/L), of which the 3 d corrosion inhibition efficiency (92.84%) was significantly higher than that of 14 d (70.38%). For this phenomenon, we believed that 14 d had a longer run time than 3 d experiment, so the nutrient-free state under the prolonged time caused death or inactivation of our B. cereus inoculum and ultimately led to a lower corrosion inhibition efficiency.
Electrochemical measurements
The Nyquist and Bode plots of Q235 CS during 14 d of rotation tests under different bacterial groups were described in Fig. 3. In general, during the first 4 days of the experiments, Nyquist plots semicircle radius in biotic group were obviously larger than that in abiotic group, reflecting that adding B. cereus inoculum significantly enhanced the Q235 CS corrosion resistance in first 4 days. Also, the radius showed different tendencies for two groups. In the abiotic group (Fig. 3a), the immersed Q235 CS gave an unoptimistic corrosive condition, the radius of Nyquist plot continuously declined over the 14 d experiments, corresponded a continuously accelerated corrosion rate. However in biotic group, Q235 CS coupons experienced a first inhibited, then accelerated corrosion rates. As Fig. 3c showed, the Nyquist plots exhibited an enlarged semicircle radius in the first 4 days, the maximum plot radius emerged in 1 d indicated the highest corrosion resistance appeared at 1 d after the 1000 mg/L B. cereus inoculum inoculated. Then in 2–4 d, the radius were slightly reduced, but still larger than 8 h. In the following 7–14 d, the radius of Nyquist plots dropped dramatically (Fig. 3c illustration), implying that B. cereus inoculum had lost its most corrosion protective function.
Table 1
Electrical elements data obtained from the best fitting of experimental impedance diagrams.
Group | Rotation Time | Rs/Ω·cm2 | Ydl/ Ω− 1sn cm2 | ndl | Rct/Ω·cm2 |
Aboitic group | 8 h | 139.9 | 0.0017765 | 0.43529 | 3160 |
1 d | 151.8 | 0.0036234 | 0.45043 | 1858 |
2 d | 186.0 | 0.00018805 | 0.66106 | 1570 |
4 d | 224.5 | 0.00030153 | 0.67683 | 1267 |
7 d | 195.9 | 0.0004017 | 0.73494 | 1042 |
10 d | 241.8 | 0.00079219 | 0.46862 | 1101 |
14 d | 139.1 | 0.00084999 | 0.58902 | 814.4 |
B. cereus inoculum group | 8 h | 146.7 | 0.00085672 | 0.73911 | 16863 |
1 d | 119.6 | 0.00015756 | 0.83567 | 20961 |
2 d | 117.3 | 0.00052107 | 0.82921 | 17403 |
4 d | 104.7 | 0.00055848 | 0.74723 | 16130 |
7 d | 169.7 | 0.00077832 | 0.67441 | 1419 |
10 d | 160.8 | 0.00069282 | 0.68932 | 968.8 |
14 d | 237.0 | 0.0046956 | 0.35044 | 961.6 |
The corresponding Bode plots were showed in Fig. 3b and d. The phase angle of Bode plots showed only one peak at low frequency in our experiments, which attributed to the formation of a passive film on the surface of the working electrode (Giorgi-Pérez et al., 2021). The larger phase ankle values, the better protection (Dong et al., 2020). Furthermore, a corresponding equivalent circuit was designated for this transfer charge process as shown in Fig. 3e, where Rs represents the solution resistance, Rct is the charge transfer resistance at the interface of substrate/film, the capacitance for electrical double layer at the metal/solution interface is denoted by Qdl. The EIS results were fitted using Zview and shown in Table 1. It can be seen from Table 1 that the impedance of Rs fluctuated with immersion time, but the Rct of biotic group exhibited a first rising and then falling trend. The Rct first rose to a maximum value of 20,961 (Ω·cm2) at 1 d and then gradually decreased to only 961.6 (Ω·cm2) at 14 d, demonstrating the first inhibited and then accelerated corrosion of Q235 CS coupons in the presence of 1000 mg/L B. cereus inoculum. In contrast, Rct values of abiotic medium were far lower than in biotic medium, which kept decline trend from 3160 to 814.4 (Ω·cm2) throughout the experiments, confirming the continuous accelerated corrosion rates in abiotic group.
Figure 3f and g displays the potentiodynamic polarization curves of the Q235 CS during 14 days of rotation in the abiotic and biotic group. The corresponding parameters are listed in Table 2. As can be seen, Q235 CS coupons in biotic medium had lower corrosion current densities (Icorr) compared to the coupon in abiotic medium. This suggested that B. cereus protected Q235 CS from corrosion to a certain extend. The Icorr of this 2 groups gradually increased during the experimental cycle, indicating that the corrosion condition was gradually deteriorating from 1–14 d, which is consistent with the results of EIS.
Table 2
Polarization data of Q235 CS after 14 days test in 14 d rotation tests.
Group | Rotation Time | Ecorr (V vs SCE) | Icorr (µA cm− 2) | βc (V dec− 1) | βa (mV dec− 1) |
Aboitic group | 1 d | -0.622 | 77.7 | 1.59 | 6.383 |
2 d | -0.654 | 885.3 | 2.131 | 8.687 |
4 d | -0.628 | 924.6 | 2.756 | 9.316 |
7 d | -0.582 | 974.0 | 2.345 | 6.349 |
10 d | -0.621 | 118.4 | 2.371 | 6.202 |
14 d | -0.63 | 161.2 | 1.315 | 4.808 |
B. cereus inoculum group | 1 d | -0.543 | 64.2 | 1.732 | 9.919 |
2 d | -0.685 | 71.7 | 2.275 | 8.547 |
4 d | -0.631 | 80.3 | 2.726 | 6.245 |
7 d | -0.657 | 93.7 | 2.103 | 7.718 |
10 d | -0.471 | 103.1 | 2.761 | 5.554 |
14 d | -0.624 | 106.4 | 2.153 | 6.973 |
Surface morphology observation
SEM along with EDS analysis
The surface morphology and elemental composition of tested Q235 CS were analyzed by SEM-EDS analysis. Surface morphology of pristine Q235 CS without immersion is displayed in Fig. s4, it showed a regular vertical strips of texture and elemental composition of Fe (92.37%) and C (7.63%).
Figure 4 presents the surface morphologies and EDS results of Q235 CS coupons in rotation tests inoculated with B. cereus inoculum. Among which, Fig. 4a, b, and c respectively represents Q235 CS after 1 d, 7 d, and 14 d rotations. It can be seen from Fig. 4a that a large number of B. cereus bodies were densely attached to the Q235 CS surface, indicating that B. cereus could complete the attachment to metal surface within 1 d after addition, similar morphologies of B. cereus was also found in a previous study (Aïmeur et al., 2015b; Qu et al., 2015). In addition, some crystal particles were intertwined around some bacteria, the EDS results revealed location of crystal particles (point 1: O 34.05%, Fe 46.12%) had an increase in O ratio and a decrease in Fe ratio compared to location of bacteria (point 2: O 19.58%, Fe 65.83%), indicating more oxidation reactions occurred at the location of crystal particles, while the corrosion due to iron oxidation may less severe at the location where the bacteria attached. At 7 d in Fig. 4b, the surface of Q235 CS distributed mainly dense homogeneous and agglomerated spherical crystals, which were identified as iron oxides in the EDS pattern (point 2). Some slender strips were attributed to bacterial metabolites due to a relatively higher C content (point 1: C 5.73%), while Bacteria is no longer visible on the surface. At 14 d in Fig. 4c, non-uniform corrosion products appeared on the surface and the Fe element content had decreased compared with 1 d and 7 d. Upper the layer of iron oxides emerged agglomerates of massive materials, which contained higher proportion of organic elements than 1 d and 7 d. Point 1 had 22.37% and 36.43% of C and O elements while point 2 had 17.82% and 37.68% of C and O elements, respectively, it should be organic matters with respect to microbial metabolism (Liu et al., 2017).
EDS line and mapping results of Q235 CS coupons after 1 d, 7 d, and 14 d rotation tests inoculated with B. cereus inoculum are displayed in Fig. 5. The Fe, Ca, C, and O elements were observed to be the major elements of the corrosion products. At 1 d, the O, P, and Na elements were more dense on the surface of Q235 CS in the region where the bacteria adhered, while the Fe element were relatively sparse. At 7 d, the morphology of the Q235 CS surface could be divided into two halves. On left side of Fig. 5b, Fe, O, and P elements were more concentrated, while Ca and C elements were more separated. The stronger peaks of O and Fe elements proved the presence of iron oxides. However, the right side was on the contrary, where Ca was find as the most dense element, accompanied by a certain amount of O element and a very low intensity of Fe element. The clear hexagonal rhombic crystals obtained are typical morphology Ca scale precipitates implies mineralization had occurred (Duan et al., 2008).
Figure 5c represents the EDS analysis of 14 d Q235 CS surface, the surface showed surrounded iron oxides with an enrichment of Ca element in the center, where a laminar stacking morphology could be recognized. In addition, very low content of other elements were observed, EDS mapping and line results demonstrated the elements C, O, P, and Ca were mainly rich in cluster-like material around Fig. 5c, which should be the product of microbial metabolism along with biomineralization-induced calcium scale precipitation.
Biofilm observation by CLSM
CLSM studies were carried out to examine the bacterial attachment and biofilm growth on the Q235 CS surface exposed to simulated cooling water medium. The biofilm samples on the Q235 CS surfaces at three different times behaved differently. The dense surface covered with shiny green color cells at 1 d indicated the complete attachment of B. cereus cells (Fig. 6a). At 7 d, the surface exhibited aggregated green fluorophores with slightly darker fluorescence intensity compared to 1 d. At 14 d, the biofilm at 14 d became more heterogeneous with green fluorescence gathered as clusters. The green fluorophore intensity became weakened once again, appeared that the biofilm on Q235 CS surface had been decayed. The clusters were also found in the SEM results (Fig. 4c and Fig. 5c), which demonstrated that at later stage of the experiment, the biofilm on Q235 CS surfaces existed mainly as a aggregated clusters rather than compact bacteria covered at 1 d.
16s analysis along with water quality indexes
The changes in bacterial community structures in 14 d of rotation tests are monitored by 16S rRNA gene amplicon sequencing. Bacteria composition at genus level are compared in Fig. 7a for 1 d, 7 d, and 14 d, respectively. At 1 d, Acinetobacter (36.2%), uncultured_bacterium_f_Enterobacteriaceae (25.8%), Paenibacillus (9.9%), and others (15.2%) were the dominated genera. However when ran to 7 d, the main microbial community structure changed significantly, the Azospirillum accounted for the most (61.3%), other strains showed varying degrees of decline. The changes in Azospirillum are closely related to ammonia nitrogen in water. Azospirillum as a well-known nitrogen fixing bacterium, ammonium, nitrate, nitrite, amino acids and molecular nitrogen can all serves as its nitrogen sources (Steenhoudt and Vanderleyden, 2000). The increase in its relative abundance could be caused by the death of dosed B. cereus released organic nitrogen acted as its energy sources. Finally at 14 d, the bacteria in simulated cooling water was mostly composed of miscellaneous bacteria from the environment, Pseudoxanthobacter (15.6%), uncultured_bacterium_p_Armatimonadetes (12.1%), possible_genus_04 (8.8%) and others (42.4%) were found to be more abundant. The B. cereus inoculum we dosed probably belongs to the genus Paenibacillus, it occupied a relative low abundance of 9.9% at 1 d, 1% at 7 d, and 0.5% at 14 d, respectively. This may be due to the lack of nutrients and the open experimental environment of simulated cooling water, where B. cereus inoculum easily be defeated by environmental microorganisms thus could not occupy a dominant population position.
Figure 7b and c displayed changes of four water quality indexes over time, pH value, DO, NH3-N, and COD. During the 14 d rotation tests, pH and DO values showed continuous increasing trends (Fig. 7b). pH values increased from 5.5 at 1 d to 7.5 at 14 d, which probably due to the hydrogen precipitation reaction produced certain amount of OH−(Fig. 7a) (Qu et al., 2017)(Qu et al., 2015). DO value maintaining around 0 mg/L for the first two days and kept continuous increasing in the rest period. The 0 mg/L DO value in 2 d is closely related to the metabolic of dosed B. cereus inoculum (Suma et al., 2019), and the death of which led to a subsequent increasement in DO. Finally, The DO value reached 3.09 mg/L at 14 d, which is disadvantageous for Q235 CS corrosion protection.
The changes of NH3-N and COD are shown in Fig. 7c. NH3-N values increased 5.60 to 6.85 mg/L from B. cereus inoculum dosed to 1 d, this slight increase was because of fractionation of residual substrates. The NH3-N values then decreased from 6.85 to 4.33 mg/L in 3 days, probably due to the degradation by our dosed B. cereus inoculum. As it has been widely accepted that B. cereus able to reduce ammonia nitrogen (Hlordzi et al., 2020). In the following 4 days, the NH3-N values continually increased, reached a maximum value of 10.43 mg/L at 6d. This presumably attributed to the inactivation and breakdown of our activated B. cereus inoculum (brown sugar + urea). There are some microorganisms have the ability of decomposing organic nitrogen to produce ammonia, such as Azospirillum, a free living N bio-fixer, which is capable of synthesizing molecular nitrogen into the ammonia state (Steenhoudt and Vanderleyden, 2000). The previous 16s sequencing results had indicated an increasement on its relative abundance in between 1 d and 7 d (Fig. 7a), which can cause an distinct increase in NH3-N. The NH3-N showed decline in the last experimental period, indicated that the organic state of NH3-N had been gradually degraded by other environmental microorganisms.
The COD in cooling water showed a decreasing trend from 391.4 to 431.4 mg/L in the first 2 d, this may also due to increase in reducing substances induced by residual substrates decomposition. In the remaining tests, COD values kept decline from 431.4 to 61.7 mg/L, indicating that the concentration of reducing substances in the water body gradually decomposed by environmental microorganisms (Chaudhry et al., 2022).
Surface characterization
The compositions of the Q235 CS corrosion products after 14 d immersion with and without B. cereus inoculum were examined by XRD, FTIR and XPS. The XRD spectra in Fig. 8a demonstrates that in abiotic group, the corrosion products were mainly FeO(OH), Fe3O4 and a little Fe2O3. While under the presence of B. cereus inoculum, corrosion products were mainly FeO(OH) mixed with small amount of Fe3O4, Fe2O3 and CaCO3. The proportion of Fe3O4 was obviously reduced and it appeared to have peaks corresponding to CaCO3. This results indicated B. cereus cells adhered to metal surfaces may induce calcium carbonate precipitation.
Figure 8b-c represent the FTIR spectra of Q235 CS corrosion products after 14 d rotation tests with and without B. cereus inoculum. In Fig. 8b, broad peaks at 3149.75 cm− 1 were attributed to C-H and N-H groups related to surface adsorption of water molecules or organic matter such as proteins and polysaccharides (Li et al., 2019). The peak at 1790.23 cm− 1 was related to stretching modes of Fe-EPS complex (Ghafari et al., 2013) while peaks at 1018.87 and 572.47 cm− 1 were assigned to FeO(OH) and Fe-O (Li et al., 2019). The unique fingerprints which containing peaks at 1480.13 and 858.55 cm− 1 associated with calcite structure (Santos et al., 2021). The asymmetric stretching vibration of the C-O-C group from carbohydrates was demonstrated by the peak at 1145.41 cm− 1 (Li et al., 2019). For Q235 CS corrosion product without B. cereus inoculum in Fig. 8c, the broad peak located at 3099.94 cm− 1 were attributed to O-H groups of adsorbed water molecule. Peaks at 1022.94 and 747.4 cm− 1 were ascribed to FeO(OH) while peak at 471.95 cm-1 possibly from Fe2O3 (Jasinski and Raymond, 1988). The results were consistent with the XRD results.
Figure 8 also shows the XPS spectra analysis of O 1s, Fe 2p and Ca 2p of the Q235 CS corrosion products after 14 d rotation tests without (Fig. 8d-f) and with B. cereus inoculum (Fig. 8g-i). In sterile cooling water medium, spectra of Fe 2p was decomposed into Fe, Fe2O3, Fe3O4, FeO(OH), and FeSO4 (Khan et al., 2020). However in medium inoculated with B. cereus inoculum, except conventional iron oxides, an complex of organic matter and iron Fe(CH3C(O)CHC(O)CH3)3 had also been obtained (Liu et al., 2021). The presence of Fe was mostly due to scraping of corrosion products while FeSO4 was an impurity caused by the experimental water. In the deconvoluted O 1s spectra (Fig. 8e and h), the peaks were related to iron oxides (Fe3O4 and Fe2O3), hydrous iron oxides (FeO(OH)), adsorbed water (H2O), the bond of carbon oxygen (C-O) and organic ligands (N-O) resulted from microbial processes (Liu et al., 2017; Yaseen et al., 2019). The two peaks of Ca 2p (Fig. 8f and i) were identified as Ca 2p3/2 and Ca 2p1/2 (Ni and Ratner, 2008). Its peaks’ intensities were much stronger in the presence of B. cereus inoculum than the abiotic group, indicated inoculating B. cereus inoculum induced the calcium precipitation on Q235 CS surface.