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Surfaces, Interfaces, and Applications
Marine Bacteria Provide Lasting Anti-Corrosion Activity for Steel via Biofilm-induced Mineralization Tao Liu, Zhangwei Guo, Zhenshun Zeng, Na Guo, Yanhua Lei, Tong Liu, Shibin Sun, Xueting Chang, Yansheng Yin, and Xiaoxue Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14991 • Publication Date (Web): 18 Oct 2018 Downloaded from http://pubs.acs.org on October 18, 2018
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Marine Bacteria Provide Lasting Anti-Corrosion Activity for Steel via Biofilm-induced Mineralization Tao Liu,1† Zhangwei Guo,1† Zhenshun Zeng,1‡ Na Guo,† Yanhua Lei,† Tong Liu,† Shibin Sun,† Xueting Chang,† Yansheng Yin,*† Xiaoxue Wang*‡
†College
of Ocean Science and Engineering, Institute of marine materials science and
engineering, Shanghai Maritime University, Shanghai, 201306, China. ‡Key
Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key
Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, the South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. 1
These authors contributed equally to this work.
*Corresponding authors Email:
[email protected] (X.X.W.);
[email protected] (Y.S.Y.)
Abstract Steel corrosion is a global problem in marine engineering. Numerous inhibitory treatments have been applied to mitigate the degradation of metallic materials; however, they typically have a high cost and are not environmental-friendly. Here we present a novel and “green” approach for the protection of steel by a marine bacterium Pseudoalteromonas lipolytica. This approach protects steel from corrosion in seawater via the formation of a biofilm followed by the formation of an organic-inorganic hybrid film. The hybrid film is composed of multiple layers of calcite and bacterial extracellular polymeric substances, exhibiting high and stable barrier protection efficiency and further providing in situ self-healing activity. The process involving the key transition from biofilm to biomineralized film is essential for its lasting anti-corrosion activity, which
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overcomes the instability of biofilm protection on corrosion. Therefore, this study introduces a new perspective and an option for anti-corrosion control in marine environments.
Keywords: Pseudoalteromonas lipolytica; biomineralization; anti-corrosion; steel; marine environment Introduction Steel corrosion in marine environments often results in enormous economic loss and hazardous safety conditions for ships and engineering structures.1 Strategies to control steel corrosion have mainly focused on surface modification by organic coatings,2,3 corrosion inhibitors and cathodic protection.4 However, the application of these conventional protection solutions has been hindered by limitations, such as high cost, difficulties in construction and maintenance, and degradation of organic polymers.5 Additionally, some of these treatments consume extra energy6 (e.g., imposed current cathodic protection) and cause environmental contamination. Nature presents forms of stable and lasting uncorroded surface through biofilm-induced mineralization in marine environment, a process of synthesis of inorganic mineral-like materials by living organism.7 Mollusk shells, composed of calcium carbonate bonded by a thin layer of biopolymer, are a typical example.8 However, the possibility of using shell-inspired film as a surface coating to control steel corrosion in marine environments has rarely been explored. Steel or metal surfaces submerged in the marine environments are rapidly colonized by various marine microbes.9 Initial colonization can then trigger the formation of biofilms, which are complex assemblage of microbial cells encapsulated within extracellular polymeric substance (EPS) matrix.10 Bacterial biofilms play an significant role in nutrient recycling and biodegradation of organic matter in marine environment. Deleterious effects including biocorrosion and biofouling have also been attributed to biofilm formed by marine microbes. For example, colonization of steel surfaces and subsequent biofilm establishment by corrosion-causing bacteria can increase the rate of
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corrosion of steels by 1000–10,000 times.11 In contrast, inhibitory effects of biofilm on metal corrosion have been also reported. In recent years, there is increasing interest to use biofilm-forming bacteria to reduce corrosion. The inhibitory mechanisms are involved with the depletion of oxygen on metal surfaces,12 secretion of antimicrobials to inhibit corrosion-causing bacteria13 or secretion of biosurfactants as diffusion barriers.14 However, given that biofilm formation is a highly dynamic biological process and detachment of biofilm can occur when conditions changes, the impact of biofilm on anti-corrosion processes will be difficult to predict and control over time.15 For example, the inhibitory action of biofilm can also be reversed to impart corrosive action due to the instability of the biofilm.16 The complex behavior of biofilm on steel corrosion remains largely undefined and unexplored, thus hindering the practical use of biofilm to control corrosion.15 Therefore, application of material science and molecular biology is needed to develop an understanding of these complex phenomena. Pseudoalteromonas is an important bacterial genus and is commonly found in association with abiotic and biotic surfaces in the ocean.17 Most Pseudoalteromonas have the capacity to colonize marine surfaces by producing EPS and forming biofilms,18,19 thus having great potential for the bio-control of corrosion in marine environments. Within a biofilm population, genotypic variation often occurs through mutation and selection due to the physiological heterogeneity inside biofilm community.20 Genetic diversity has been proposed as a beneficial feature of biofilms and generates a diversified population with specialized functions.21-24 Recently, we found that the genetic variants with alteration in EPS production isolated in Pseudoalteromonas lipolytica biofilm affect the larval settlement of the mussel Mytilus coruscus.25,26 Herein, two EPS variants of P. lipolytica, strain with exopolysaccharide cellulose overproduced (EPS+) and strain lacking cellulose production (EPS−) were selected to investigate the key factor effecting steel corrosion inhibition in seawater. These isogenic P. lipolytica strains exhibit very distinctive effects on steel corrosion. Morphology and composition analysis revealed that the EPS+ strain provided stable anti-corrosion activity on the steel surface, and this action was dependent on the formation of an organic-inorganic hybrid film. A novel and “green” approach for the protection of steel based on biofilm-induced mineralization was thus developed. Additionally, the hybrid film provides in situ self-healing activity. Therefore, this study
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provides an interesting insight to the use of microbial-influenced mineralization to address the instability of biofilm protection. Materials and Methods Construction of the EPS− strain. The EPS− strain of P. lipolytica (with the bcsZ and bcsB genes deleted) was constructed using our recently developed gene deletion method
for
marine
Pseudoalteromonas
strains.
The
recombinant
plasmid
pK18mobsacB-ery-bcsZB used for deleting the bcsZ and bcsB genes was constructed in our previous study.26 Briefly, this recombinant plasmid was mobilized to WT P. lipolytica through conjugation, and erythromycin (25 μg/mL) was used to select the strain with this plasmid integrated in the host genome. Sucrose counter-selection was then used to generate the EPS− strain. PCR and DNA sequencing confirmed the mutant strain. Biofilm assay. For the attached biofilm assay, overnight cultures of P. lipolytica strains were diluted to an initial OD600 of 0.1 and cultured statically in 96-well polystyrene plates (Corning Costar, Cambridge, MA, USA). The attached biofilm was stained using crystal violet as previously described.24 Normalized biofilm was measured by dividing the total biofilm by the maximal bacterial growth, as measured by turbidity at 620 nm for each strain. For pellicle biofilm formation, cultures with the initial OD600 of 0.1 were incubated statically in 2216E medium at 25°C. Pellicle formation in the liquid– air interface was visualized and imaged during culturing. The biofilm assay was repeated at least two times with three independent cultures of each strain. P. lipolytica strains were cultured in 2216E medium overnight at 25°C. Ten microliters of the cultures were plated on SWLB agar containing 1 mM calcium chloride or magnesium chloride. The colony morphology on the agar plate was visualized and imaged using the stereoscopic microscope after 5 days at 25°C. At least three independent experiments were conducted and evaluated. The steel immersion test. Steel used in this study was provided by Baosteel Company (Shanghai, China). The alloy elements composition of the steel is (wt%): 1.5% Mn, 0.70% Ni, 0.20% Si, 0.15% Ti, 0.04% Al, 0.02% Nb, 0.055% C, and balanced Fe.
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For the chemical composition and morphology analysis of the steel surface during the immersion test, a 1 cm × 1 cm steel coupon was used. For the electrochemical analysis, the steel specimens were sealed with epoxy resin. An exposure area of 1 cm × 1 cm. was leaving. All steel specimens were mechanical polished sequentially with emery papers from 200 to 800 grit. The specimens were then washed with alcohol, DI water, and were dried in N2 environment. These steel coupons were sterilized using ultraviolet (UV) light prior to use in the immersion test. The steel immersion tests were performed in 1000 mL containers with steel coupons submerged in 600 mL marine broth (2216E) or artificial seawater as shown in Figure S1. The three isogenic P. lipolytica strains (WT, EPS+ and EPS−) were cultured in marine broth (2216E medium) on a rotary shaker at 150 rpm. Approximately 0.25 mL of overnight culture of each strain was added to the container (final bacterial density ~ 5 × 106 CFU /mL), which were incubated in a shaking incubator at 150 rpm up to 14 days or 2 months at 36°C or the other temperatures (15, 20, 25°C). A control group was included with no bacteria added. Half of the medium was replaced with fresh medium every 5 days. For the corrosion rates measurement, weight loss of the immersion test was performed for 30 days. Marine broth (2216E) was purchased from Difco (BD, New Jersey, USA) and DI water was added and autoclaved prior to use. Artificial seawater purchased from Xinyuan Mining Company (China) and DI water was added and autoclaved prior to use. Morphology characterization of films on the steel surface. To check the biofilm, A LIVE/DEAD Biofilm Viability kit (Invitrogen, Thermo Fisher Scientific, USA) was used for fluorescent staining after immersion in marine broth after 5 days. The damaged membranes (dead) and intact membranes (live) showed red and green color, respectively, which were differentiated by the propidium iodide and SYTO 9 contained in the kits. In addition, partially damaged/dead cells were yellow. To characterize the morphology of the films after 14 days, the steel coupons were taken out of the containers, washed with a phosphate buffered solution (PBS, pH = 7.4), immobilized in 2.5% glutaraldehyde
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solution for 4 h. The coupons were then dehydrated sequentially in the 20%, 40%, 60%, 80%, 90%, and 100% ethanol solution for 10 min and N2 dried. The top view and cross-sectional surface were observed with a SEM (JEOL JSM-7500F, Japan). Characterization of the biomineralized film formed by EPS+. To determine the crystal structure of the biomineralized film, Atomic Force Microscopy (AFM; Dimension icon scanasyst, Bruker) analysis was carried out on the calcite crystals. A scanning frequency was 0.977 Hz and the area scanned was 600 × 600 nm. We used the ScanAsyst mode with a Si3N4 tip under a spring constant of 0.4 Nm−1 at 25°C. Furthermore, the bacteria induced minerals were analyzed by a high-resolution transmission electron microscope (HRTEM, JEM 2100F). Prior to analysis, 0.1 ml of 14 day old testing marine broth with EPS+ strain was placed on a copper grid. Selected area electron diffraction (SAED) patterns data were collected from a circular region approximately 0.2 μm in diameter using a 10 μm aperture. The phase identification of the biomineralized film was analyzed by XRD using a PANalytical X’ Pert PRO XRD, Netherlands, at 40 kV, 10 mA, with a Cu-Kα radiation source, and scanning at a rate of 0.26°/s within the 2θ range of 20 to 90°. The crystals composition was analyzed using an Infrared Raman spectrometer (Vertex 70, Bruker, Germany) in the range of 400–4000 cm−1. Electrochemical and weight loss measurements. Electrochemical impedance spectroscopy (EIS) was performed using an AutoLab electrochemical workstation (Metrohm, Switzerland). A standard 3-electrode system contained the steel specimen as the work electrode, a platinum (Pt) sheet and a saturated calomel electrode (SCE) as the counter electrode and reference electrode. The EIS was performed in the marine broth at the OCP between 10−2 and 105 Hz frequency. The sine wave disturbance amplitude was ± 10 mV. The software program Zsimpwin was used to fit the impedance data. The corrosion rate was calculated by the following equation: Vcorr = (87600 × ∆m)/ρAt
(1)
where Vcorr, ρ, A, t and ∆m are the steel corrosion rate (mm·y−1), coupon density
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(g·cm−3), exposed area (cm2), immersion time (h) and weight loss (g), respectively. Evaluation of the pitting corrosion and efficacy of self-healing. To test the pitting distribution on the steel surface, the corrosion products and attached biofilm on the coupons were removed via washing sequentially in dibutylthiourea-HCl solution, NaHCO3 solution, DI water, and acetone for 2 min each step. The morphology of the corroded steel was detected using an optical profilometer (Bruker ContourGT, USA). The local impedance in the presence of an artificial defect of 2 mm × 1 mm on the steel surface was analyzed using a localized scanning electrochemical workstation (Bio-logic M470, France) before and after 14 days of immersion with different strains. A 3-electrode contained a coated sample as the working electrode, saturated calomel electrode and a carbon rod as the reference electrode and the counter electrode, respectively. The distance between the steel surface and a 5-μm tip of Pt microprobe was adjusted to approximately 100 μm. The measurements were carried out with 10 μA current amplitude at a single frequency of 10 Hz. To ensure accuracy, 5 mM NaCl solution with a low electrical conductivity was used as the testing electrolyte. In order to verify the self-healing performance by the EPS+ strain, a 3D optical profilometer (Bruker ContourGT, USA) was also used on the biomineralized film in the presence of an artificial defect (400 μm in width, 30 μm in depth) before and after 7 days immersion with the EPS+ strain. Results P. lipolytica strains exhibit distinctive effects on steel corrosion. Among twelve Pseudoalteromonas spp. collected from various marine habitats, the surface water derived non-pathogenic strain, Pseudoalteromonas lipolytica,28 exhibited a high capacity to form biofilms at varying temperatures ranging from 10 to 37°C. Therefore, the P. lipolytica strain was selected to explore the effect of biofilm on steel corrosion in the ocean. A steel immersion test was first conducted by inoculating the WT strain in marine broth (2216E medium) with a submerged steel coupon, and the steel corrosion was monitored over time at 36°C (Fig. S1). For the control group (marine broth only), corrosion occurred at the steel surface after 1 day of immersion with the formation of
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uneven ferric precipitates. The surface continued to rust to day 14. In contrast, WT showed strong anti-corrosion activity, but signs of corrosion occurred after a long immersion time (after 14 days) (Fig. 1A). To further evaluate the correlation between biofilm formation and corrosion inhibition, two mutants with increased and reduced biofilm formation were chosen for further study. The EPS+ strain was an exopolysaccharide cellulose overproducing strain that was identified during the screening of genetic variants in P. lipolytica biofilms from our earlier study.26 The EPS− strain lacking cellulose production was constructed by knocking out the cellulose cluster bcs in P. lipolytica. When grown in marine broth, all three strains have the ability to form attached biofilm at the solid–liquid interface or “pellicle” biofilm at the liquid–air interface (Fig. 1BC). The EPS+ strain also developed into a well-organized wrinkled “colony biofilm” at the solid-air interface after long incubation in seawater LB (SWLB) agar plate, further developing into a more compact “colony biofilm” with the addition of 1 mM Ca2+. In contrast, the WT or EPS− strain formed a smooth “colony biofilm” at the solid-air interface with and without the addition of Ca2+ or Mg2+ (Fig. 1D). Thus, the EPS+ strain had greater ability to form biofilm than the WT strain or the EPS− strain at various interfaces. The two mutants were then tested for their ability to control steel corrosion. As expected, the EPS− strain formed ferric precipitates on the steel surface on day 1, which persisted to day 14, providing no protection of the steel surface. In contrast, the EPS+ strain demonstrated the strongest anti-corrosion ability and showed no signs of corrosion after 14 days (Fig. 1A). More importantly, the EPS+ strain provided stable anti-corrosion protection for 2 months, while numerous pits were observed on the steel surface for the WT strain under the same tested conditions (Fig. S1A). Similarly, when artificial seawater was used, the EPS+ strain could still protect the steel coupons from corrosion after a long immersion time, while the WT and EPS- strains did not show stable anti-corrosion activity (Fig. S1B). In addition, a LIVE/DEAD Biofilm Viability kit was used to assess the viability of bacterial cells on the steel surface submerged in marine broth using CLSM on day 5. In the mixed population, bacterial cells with intact/damaged cell membranes stain fluorescent green and red, respectively. As shown in Fig. 1E, all three strains formed biofilms on the steel surface on day 5. The majority of the bacterial cells in the WT biofilm remained alive and these cells were distributed evenly in the
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biofilm (~10 μm in depth). A similar pattern was observed for the EPS− biofilm, except that more dead cells were present in the EPS− biofilm (~50 μm in depth). In contrast, the live cells of the EPS+ strain were all embedded in mushroom-like structures of the biofilm (~35 μm in depth) that formed on the steel surface. Furthermore, the dissolved oxygen concentration in the marine broth inoculated with the three isogenic P. lipolytica strains was measured during the immersion test (Fig. S2). As expected, the level of dissolved oxygen inoculated with the P. lipolytica strains was close to zero and less than the control group at an early stage (day 1 to 5), suggesting that these strains all consumed oxygen via respiration at this stage. However, the ability to consume dissolved oxygen was significantly reduced in the EPS+ strain compared to the EPS− strain at later stages (e.g. day 6 and 7), suggesting that oxygen depletion through respiration by live cells within the biofilm was not the primary reason for corrosion reduction by the EPS+ strain. These results demonstrate that the mutants of P. lipolytica had distinctive effects on steel corrosion, and the inhibitory action of the EPS+ strain was not attributed to oxygen depletion through bacterial respiration during biofilm formation. Biofilm formed by the EPS+ strain induces biomineralization on the steel surface. To probe the surface morphology in the presence of different P. lipolytica strains, scanning electron microscopy (SEM) was performed on day 14 of the immersion test (Fig. 2). In the control group, rough and cracked corrosion products were formed on the steel surface, and the magnified view displayed a typical petal-like Fe3O4 structure (Fig. 2AB). When inoculated with the WT strain, a rough film was formed on the steel surface (Fig. 2C). The magnified view shows that the bacterial cells were entangled with EPS to form scale-like structures on the steel surface (Fig. 2D), indicating the steel was corroded though no visible sign of corrosion was observed (as shown in Fig. 1A). Surprisingly,
a
relatively
dense
mineral-like
film
featured
as
trigonal
or
quasi-rhombohedral structures was formed on the steel surface for the EPS+ strain (Fig. 2E). The magnified view revealed that each trigonal or rhombohedral structure was formed by layers of small trapezoidal pillars growing in the same direction (Fig. 2F). Few bacterial cells were also found that aligned with the direction of the trapezoidal
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pillars. As for the EPS− strain, dense biofilms, along with abundant bacterial cells and corrosion products rather than mineral precipitates, covered the steel substrate (Fig. 2G). The SEM image under high magnification clearly showed loose corrosion products overlapped the EPS− cells (Fig. 2H). X-ray diffraction (XRD) was then used to determine the composition of films by different P. lipolytica strains. As shown in Fig. S3A, the main components on the steel surface of the control group were Fe and Fe3O4. Similarly, Fe also comprises the main components for the groups containing the WT and EPS− strains. In contrast, XRD shows that (Mg0.064Ca0.936) (CO3) was the main component of the mineral precipitates on the steel surface containing the EPS+ strain, which had 3 major diffraction peaks of 29.5°, 48.7° and 47.7°, which has been reported earlier for calcium carbonate.29 These results suggest that the EPS+ strain might induce calcium carbonate precipitation to form calcite on the steel surface. The presence of calcite was further confirmed with Fourier transform infrared (FTIR) spectroscopy (Fig. S3B). A unique fingerprint of spectrum can be identified as calcite, containing a ν2 peak at 876 cm−1, ν3 peak at 1426 cm−1 and ν4 peak at 712 cm−1. The 1426 cm−1 vibration peak was not as sharp as the other peaks due to the overlap of the vibration of organic matter and inorganic mineral.30 Furthermore, vibration peaks of organic matter were also observed in the infrared spectra. The peak at 1690 cm−1 represented the C=O vibration from carboxylic acid. The C-H stretching vibration of organic matter was observed between 2800–3000 cm−1.31 The 3429 cm−1 peak was due to the O-H bond. These results suggest that this biomineralized film is an organic-inorganic hybrid film that is composed of organic matter and calcite. To elucidate the architecture of the biomineralized film formed by the EPS+ strain, cross-section SEM and atomic force microscopy (AFM) imaging were performed at multiple locations on the steel sample on day 14. Figure 3A demonstrates the thickness of the biomineralized film on the steel surface was approximately 43 μm, and the interface between the film and substrate was closed and compact. Magnified SEM images
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corresponding to AFM images of the calcite crystal details were also performed on day 14. The calcite crystal exhibited trapezoidal pillars with a hierarchical structure (Fig. 3B) and the size of each pillar was approximately 600 nm in length and 300 nm in width (Fig. 3C). TEM analyses of the EPS+ cell showed that nanometer-sized precipitates (~200–400 nm in diameter) were embedded in the EPS, which was attached to the bacterial cell wall (Fig. 3D). These nanometer-sized precipitates were identified as a mixture of polycrystalline and amorphous structures according to the SAED pattern, which showed clear diffraction spots and fuzzy diffuse rings (Fig. 3E). High-resolution TEM (HRTEM) analyses show that the precipitates were polycrystalline with at least three planes. The lattice spacing of the precipitates was 0.25 nm, 0.30 nm, and 0.38 nm, corresponding to the (110), (104), and (012) planes, respectively (Fig. 3F). Consistent with the XRD analysis, the lattice spacing pattern further confirmed that the precipitates were calcium carbonate precipitations.29 Calcite formation requires three major conditions: pH, a nucleation site, and Ca2+ and/or Mg2+.30 EPS can complex Ca2+ and/or Mg2+ ions, which provides a site for nucleation as a start of precipitation.32 Our analysis suggested that precipitation started near the bacterial cells, presumably in the EPS and calcium carbonate precipitations formed on the steel surfaces only for cells overproducing EPS (EPS+ strain). In addition, when tested by “colony biofilm” formation in solid agar plates, the addition of excess Ca2+ significantly changed the matrix morphology of the EPS+ strain but not for the WT or EPS− strains (Fig. 1D), indicating that only the EPS+ strain can effectively bind Ca2+. These results suggest that the EPS+ strain can induce the formation of biomineralized film on the steel surface, which is an organic-inorganic hybrid film that composed of biofilm (bacteria and EPS) and calcite. Biomineralization by EPS+ provides strong anti-corrosion protection. EIS has been widely used for the evaluation of steel corrosion processes. Thus, we used the diameters of the Nyquist plot obtained from EIS measurements to evaluate the barrier
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protection efficiency of different strains. As shown in Fig. 4A, the barrier protection efficiency of the EPS+ strain was comparable to those of the control or the other two strains at the early stage (≤5 d). In contrast, at the late stage (≥9 d), the EPS+ strain provided the strongest barrier protection efficiency among the tested groups, with the diameter of the Nyquist plot 102-fold larger than the other groups. In addition, different shapes of the Nyquist plots were attributed to the different stages of steel corrosion during the immersion test. At the early stage (≤5 d), one loop in the Nyquist plot showed only one time constant in the process of the electrochemical reaction, indicating that the compact film was not yet developed at this stage for all the strains. At the late stage (≥9 d), Nyquist plots of the EPS+ group showed an obvious upward divergence, which resulted in much higher diameters for the EPS+ group than the other tested groups. In contrast, only one loop was observed for the WT group during the whole testing time (14 d), indicating no compact film developed even after 14 days. The barrier protection efficiency of different strains was also determined using the EIS fitting results of Rp(Rf+Rct) as a function of time (Fig. 4B). The Rp values are indicative of the anti-corrosion efficiency. The EIS curves were fitted (errors EPS− > WT > EPS+, which is negatively correlated with its ability of inhibiting corrosion. It is generally known that pitting is a characteristic of steel biocorrosion. Some live cells in the biofilm could also obtain electrons directly from the steel in the absence of organic carbon, resulting in narrow pits on the steel.8 Since all three strains can form biofilm on the steel surface, we tested whether these biofilms affect the pitting corrosion of the steel surface using microscale in situ optical profile analyses (Fig. 5AB). For the control group, many pits were visible on the steel coupon, and the average pit size was approximately 22 μm in depth and 77 μm in diameter after 14 days. The size of the pit on the steel surface was also analyzed for the three different strains after removing the films after 14 days. In the case of the EPS− strain, there were deeper, larger pits (approximately 34 μm in depth and 100 μm in diameter). For the WT strain, pitting corrosion was also present on the steel, although the pits tended to be shallow and small (approximately 24 μm in depth and 7 μm in diameter). In contrast, the steel coupon induced by the EPS+ strain displayed an uncorroded surface without obvious pit corrosion. On this aspect, the pitting inhibition effect of biomineralized film was superior to cathodic protection, as the pitting corrosion still occurred on the steel under the cathodic protection potential in the presence of microorganisms. These results demonstrate that biomineralization film formed by the EPS+ strain has high impedance, which can greatly reduce the corrosion
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rate of the steel and pitting on the steel surface. EPS+ strain effectively provides in situ self-healing activity. Since coating on the steel may encounter sharp edges in the ocean that scratch the surface, we investigated whether this biomineralized film has the capacity for in-situ self-healing when a scratch is present. Thus, an artificial crack was first made on the biomineralized film formed by the EPS+ strain after 14 days and LEIS measurement was used to investigate whether this damage can be repaired using the EPS+ strain. Figure 6A shows that the local impedance in the scratched area was much less than that in the intact area in the beginning. Specifically, the average impedance value in the center of the scratched area was approximately 400 Ω·cm2, approximately 104-fold lower than the value of the intact area, suggesting that the biomineralized film in this scratched area was completely destroyed. After submerging in marine broth inoculated with the EPS+ strain for 7 days, the average impedance in the damaged area reached 6.0 × 104 Ω·cm2, which was comparable to the intact area (Fig. 6B). As expected, the sharp drop in impedance (104-fold drop) between the intact area and the damaged area was replaced with a gradual slow drop (2-fold) after 7 days. In addition, 3D optical profiles of the surface morphology were used to evaluate the self-healing performance of the biomineralized film. Figure 6C shows the morphology of the biomineralized film in the presence of artificial scratch (400 μm in width, 30 μm in depth). After submerging in marine broth inoculated with the EPS+ strain for 7 days, the scratch was mostly re-filled and the repaired area appeared similar to the intact area (Fig. 6D). These results demonstrate that the EPS+ strain provided in situ self-healing activity of corrosion control. Discussion Corrosion inhibition by biofilm has captured considerable attention in recent years. Biofilms formed by microorganisms have been found to inhibit metal corrosion processes including E. coil,11 Shewanella oneidensis,12 Bacillus spp.,10,34 Pseudomonas spp.,13,35 Pseudoalteromonas spp.36 and Vibrio neocaledonicus.37 However, due to the dynamic
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nature of biofilm that is dispersed and dissolved easily, the durability of the anti-corrosion activities of biofilms remains a significant issue. In this study, the stable corrosion protection of steel coupons by the exopolysaccharide overproducing variant (EPS+) of marine bacterium P. lipolytica was demonstrated by the steel surface morphology characterization and corrosion evaluations. In contrast, the WT and EPS− strains of P. lipolytica could also formed biofilm but were ineffective in the case of corrosion inhibition under the same tested condition. We provide evidence that biomineralization is the key process for successful inhibition of corrosion on steel surface conferred by the EPS+ strain. As a result, the instability of biofilm protection can be solved by forming an organic-inorganic hybrid via biofilm-induced mineralization before the dispersal of biofilm. The barrier protection and pitting inhibition properties imparted by this biomineralized film were comparable to or even better than some previously reported organic coatings and cathodic protection methods used for the anti-corrosion protection of steel.38 Notably, Pseudoalteromonas strains are ubiquitously distributed in the ocean39 and majorities of them, including P. lipolytica, are non-pathogenic. Therefore, this study presents a novel and “green” strategy for the protection of a steel surface from corrosion in the ocean. It has been reported that over 200 bacteria strains, including Bacillus subtilis, Pseudomonas spp., and Azotobacter spp., are also capable of inducing calcium carbonate precipitation in soil.40 Microbial influenced mineralization is a complex process that relies on the environmental physiochemical conditions and the metabolism of the microorganism. Calcium carbonate precipitation requires the nucleation site, the presence of Ca2+ and/or Mg2+, and proper pH,41 and our results showed that the pH was more than 8.0 when P. lipolytica induced the initial calcium carbonate precipitation (Fig. S2b). It has been shown that the addition of urea can generate carbonate and elevate pH via urea hydrolysis to provide necessary conditions for biomineralization in bacteria. Here, we presented evidence that marine bacterium P. lipolytica can induce biomineralization in
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seawater without the addition of urea. Furthermore, biofilms formed by the EPS+, WT, and EPS− strains of P. lipolytica exhibited rather different carbonatogenic capacities, although they all have the ability to form biofilms. It has been reported that exopolysaccharide cellulose matrices can absorb Ca2+ and Mg2+, which promote calcite crystal formation by providing additional nucleation sites.42 Consistent with this result, we found that the EPS+ strain with overproduced cellulose can bind Ca2+ and facilitate the formation of calcite in seawater, while no such action was found for the WT or EPS− strains. Unlike other biofilm-induced precipitation, as shown by Pseudomonas aeruginosa that requires the addition of extra Ca2+ or Mg2+,43 the biomineralization induced by P. lipolytica occurred in seawater or marine broth. The microbial metabolism can promote carbonate alkalinity and accelerate subsequent calcium carbonate precipitation.30,43 Furthermore, our finding that biomineralization preferentially occurred on the EPS+ biofilm instead of the WT biofilm suggests that biomineralization might be closely correlated with the 3D wrinkled structure of biofilm, which may facilitate mineral deposition.
Future
applications
using
this
biofilm-induced
calcium
carbonate
precipitation to control corrosion on other surfaces still requires the ability of target bacteria to form biofilm and induce biomineralization under specific environmental conditions. Previous work showed that the hierarchical structure of biominerals, including elongated prismatic crystals with different orientations, strongly contributes to the reduction of Cl− ions or O2 diffusion.44 The barrier effect may explain why the EPS+ strain inducing biomineralized film displayed much higher resistance values than those of WT, EPS−, and control groups. For the WT group, the protection strategy is mainly dependent on biofilm formed at early stage. Although the biofilm is uniform and can protect the steel surface over a short period, the metabolism of the attached cells enhances the probability of pitting nucleation. As for EPS−, lacking cellulose production resulted in more serious pitting corrosion than the control group. Here, we demonstrated
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that P. lipolytica strains can either inhibit or promote metal corrosion, and this effect is highly dependent on extracellular cellulose production and the ability to induce biomineralization. To evaluate this application in an actual ocean environment with varying temperature, the ability to induce the formation of biomineralized film by the EPS+ strain was assessed with experimental temperatures at 15, 25, or 30°C. Although the temperate affects the time needed to develop the biomineralized film, the composition and properties of the biomineralized film formed by the EPS+ strain have no obvious differences. In addition, we also assessed the ability of EPS+ to form biomineralized film on the steel surface in the presence of competing marine bacteria. For this purpose, Vibrio natriegens that was considered as one of the fastest growing bacteria in ocean was added to the marine broth with EPS+ strain at equal cell numbers. Our results showed that the growth of V. natriegens was significantly inhibited by the EPS+ strain, and as a result, the biomineralized film was still formed on the steel surface by the EPS+ strain (Fig. S5). It has been reported that many Pseudoalteromonas strains produce a variety range of antimicrobial compounds,18 conferring a growth advantage for Pseudoalteromonas in a competitive marine environment and ensuring its role as one of the pioneering members in surface-attached community in various marine habitats. These features further cement the use the EPS+ strain to coat steel surfaces in ocean engineering and further genetic modification of this strain can be made in conditions needed. Conclusion In summary, we demonstrate that the transition from biofilm to biomineralized film is the key process for marine bacteria to provide stable anti-corrosion protection for steel in the marine environments. This work also introduces a new perspective for the development of stable and environmental-friendly anti-corrosion coatings on various surfaces via the biofilm-induced mineralization processes in other environments.
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ASSOCIATED CONTENT Supporting Information Figure S1.
Images of the corrosion morphology submerged in marine broth for 60 days
and artificial sea water for 14 days. Figure S2.
Measurements of pH and dissolved oxygen (DO).
Figure S3.
XRD spectrum and FTIR spectra.
Figure S4.
Bode plots and Equivalent circuit diagrams for the steel specimens.
Figure S5.
The ability of EPS+ to form biomineralized film with competing marine
bacteria. Table S1. Electrochemical impedance parameters fitted from the impedance plots. AUTHOR INFORMATION Corresponding Authors *E-mail:
[email protected] (X.X.W.). *E-mail:
[email protected] (Y.S.Y.). Notes The authors declare no competing financial interest. Acknowledgments Funding: Authors X. X. W. and T. L. received founding from National Basic Research Program
of
China
(2017YFC0506303,
2014CB643306,
2016YFB0300702,
2018YFC1406500) and the National Science Foundation of China (31625001). Author contributions: X. X. W. and Y. S. Y conceived the concept and designed the experiments. T. L. and X.X.W. drafted the manuscript. Z.W.G. and N. G. performed all corrosion characterization and data analysis. Z.S.Z. performed bioanalysis and biofilm characterization. Y.H.L. and T. L. performed TEM measurements and analyzed data. S.B.S. and X.T.C. analyzed XRD and FTIR data. All authors discussed results and analyzed data. References
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(31) Pokroy, B.; Quintana, J. P.; Caspi, E. N.; Berner, A.; Zolotoyabko, E. Anisotropic Lattice Distortions in Biogenic Aragonite. Nat. Mater. 2004, 3, 900-902. (32) Li, X.; Chopp, D. L.; Russin, W. A.; Brannon, P. T.; Parsek, M. R.; Packman, A. I. Spatial Patterns of Carbonate Biomineralization in Biofilms. Appl. Environ. Microbiol. 2015, 81, 7403-7410. (33) Anandhi, A.; Palraj, S.; Subramanian, G.; Selvaraj, M. Corrosion Resistance and Improved Adhesion Properties of Propargyl Alcohol Impregnated Mesoporous Titanium Dioxide Built-in Epoxy Zinc Rich Primer. Prog. Org. Coat. 2016, 97, 10-18. (34) Qu, Q.; He, Y.; Wang, L.; Xu, H. T.; Li, L.; Chen, Y. J.; Ding, Z. T. Corrosion Behavior of Cold Rolled Steel in Artificial Seawater in the Presence of Bacillus subtilis C2. Corros. Sci. 2015, 91, 321-329. (35) Gunasekaran, G.; Chongdar, S.; Gaonkar, S. N.; Kumar, P. Influence of Bacteria on Film Formation Inhibiting Corrosion. Corros. Sci. 2004, 46, 1953-1967. (36) Wu, J. J.; Zhang, D.; Wang, P.; Cheng, Y.; Sun, S. M.; Sun, Y.; Chen, S. Q. The Influence of Desulfovibrio sp and Pseudoalteromonas sp on the Corrosion of Q235 Carbon Steel in Natural Seawater. Corros. Sci. 2016, 112, 552-562. (37) Moradi, M.; Xiao, T.; Song, Z. L. Investigation of Corrosion Inhibitory Process of Marine Vibrio neocaledonicus sp Bacterium for Carbon Steel. Corros. Sci. 2015, 100, 186-193. (38) Guo, J.; Yuan, S. J.; Jiang, W.; Lv, L.; Liang, B.; Pehkonen, S. O. Polymers for Combating Biocorrosion. Front. Mater. 2018, 5, 10-25. (39) Wietz, M.; Gram, L.; Jorgensen, B.; Schramm, A. Latitudinal Patterns in the Abundance of Major Marine Bacterioplankton Groups. Aquat. Microb. Ecol. 2010, 61, 179-189. (40) Boquet, E.; Boronat, A.; Ramoscor.A. Production of Calcite (Calcium-Carbonate) Crystals by Soil Bacteria is a General Phenomenon. Nature. 1973, 246, 527-529. (41) Dupraz, C.; Reid, R. P.; Braissant, O.; Decho, A. W.; Norman, R. S.; Visscher, P. T. Processes of Carbonate Precipitation in Modern Microbial Mats. Earth-Sci. Rev. 2009, 96, 141-162.
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(42) Nishimura, T. Macromolecular Templates for the Development of Organic/Inorganic Hybrid Materials. Polym. J. 2015, 47, 235-243. (43) Bai, Y.; Guo, X. J.; Li, Y. Z.; Huang, T. Experimental and Visual Research on the Microbial Induced Carbonate Precipitation by Pseudomonas aeruginosa. Amb. Express. 2017, 7, 57-65. (44) May-Crespo, J.; Martinez-Torres, P.; Alvarado-Gil, J. J.; Quintana, P.; Ordonez-Miranda, J. Water Transport Monitoring in Calcium Carbonate Stones by Photoacoustic Spectroscopy. Int. J. Thermophys. 2010, 31, 1027-1036.
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Figures and Tables
Figure 1. P. lipolytica strains exhibited different effects on steel corrosion and different abilities of biofilm formation. (A) Images of the steel surface submerged in marine broth from day 1 to day 14 inoculated with three different strains. The control group contains only marine broth. (B) The formation of “attached biofilm” at the solid-liquid interface for three different strains was assessed by culturing statically in polypropylene microtitre plates in marine broth. (C) The formation of “pellicle” biofilm at the liquid-air interface three different strains was assessed by statically culturing in glass tubes in marine broth. (D) The formation of “colony biofilm” at the solid-air interface for three different strains was assessed after 5 days on seawater LB (SWLB)
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agar plate. (E) Confocal laser scanning microscopy images of the biofilm cells stained with the LIVE/DEAD Biofilm Viability kit on the steel surface on day 5 of the immersion test. Live cells are shown as green, dead cells as red and partially damaged/dead cells as yellow. Experiments were performed three times with at least three replicates of each strain and only representative images are shown in A, C, D and E. Data are from three independent cultures, and one standard deviation is shown in B.
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Figure 2. SEM images of the steel surface inoculated with different P. lipolytica strains. Image of two different magnitudes are shown in the left (A, C, E, G) and right panel (B, D, F, H) for each strain at day 14 during the immersion test, respectively. Red arrows in the enlarged images of d, f and h indicate the bacterial cells. Experiments were
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performed twice with at least three replicates of each strain and only representative images are shown here.
Figure 3. EPS+ strain induces calcite biomineralization on the steel surface. (A) The cross-sectional SEM images of biomineralized film induced by EPS+ strain on day 14. (B) High magnification SEM image of the morphology of biomineralized film induced by EPS+ strain on day 14. (C) AFM image of the biomineralized film induced by the EPS+ strain on day 14. The scanning size was 600 nm × 600 nm. (D) TEM image of the EPS+ cell in the marine broth on day 14 during the immersion test. Nanometer-sized precipitates (c) embedded inside EPS (EPS) is attached to the wall of the bacterial cell (bc). (E) Selected area electron diffraction (SAED) pattern showed that the nanometer-sized precipitates embedded in EPS were a mixture of polycrystalline and amorphous structures. (F) High-resolution transmission electron microscopy (HRTEM) analyses showed that the nanometer-sized precipitates were polycrystalline with at least three planes: (012), (104), and (110).
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Figure 4. Biomineralization by EPS+ provides strong anti-corrosion protection. Corrosion protective efficacy was evaluated for different strains of P. lipolytica over time. (A) Nyquist plots for the steel specimens at different immersion times on day 1, 5, 9 and 14. (B) The EIS fitting results of Rp(Rf+Rct) as a function of time. Error bars indicate the fitting errors. (C) The corrosion rates from a weight loss test (up to 30 d) were performed for different strains in marine broth. Data are from three independent cultures and one standard deviation is shown in C.
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Figure 5. EPS+ strain inhibits pitting corrosion. (A) Images of the optical profilometry of the pit morphology and (B) derived topography (pits diameter and depth) on the steel surface after removing the biofilm during the immersion test for three different strains. The scanning size was 950 μm × 1262 μm. Data are from three independent cultures, and one standard deviation is shown in B.
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Figure 6. EPS+ strain provides in situ self-healing effectiveness on steel surface. The electrochemical impedance spectroscopy (LEIS) (A) and three-dimensional (3D) optical profiles (C) illustrated the feature of artificial scratch made on the biomineralized film formed by the EPS+ after 14 days. After 7 days in marine broth with the EPS+ strain, LEIS (B) and 3D profiles (D) illustrated the the artificial scratch made on the biomineralized film was repaired and mostly refilled.
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