Multi-scale analysis of acidophilic microbial consortium biofilm's tolerance of lithium and cobalt ions in bioleaching.

Metal ions stress will inhibit the oxidation capacity of iron and sulfur of an acidophilic microbial consortium (AMC), which leads to reduced bioleaching efficiency. This work explored the impacts of Li+ and Co2+ on the composition and function of AMC biofilms with a multi-scale approach. At the reactor scale, the results indicated that the oxidative activity, the adsorption capacity, and the biofilm formation ability of AMC on pyrite surfaces decreased under 500 mM Li+ and 500 mM Co2+. At the biofilm scale, the electrochemical measurements showed that Li+ and Co2+ inhibited the charge transfer between the pyrite working electrode and the biofilm, and decreased the corrosion current density of the pyrite working electrode. At the cell scale, the content of proteins in extracellular polymers substrate (EPS) increased as the concentrations of metal ions increased. Moreover, the adsorption capacity of EPS for Li+ and Co2+ increased. At the microbial consortium scale, a BugBase phenotype analysis showed that under 500 mM Li+ and 500 mM Co2+, the antioxidant stress capacity and the content of mobile gene elements in AMC increased. The results in this work can provide useful data and theoretical support for the regulation strategy of the bioleaching of spent lithium-ion batteries to recover valuable metals.

Corrosive Pseudomonas aeruginosa detection by measuring pyocyanin with a lab-on-fiber optical surface plasmon resonance biosensor in aquatic environments.

Oceanic facilities and equipment corrosion present considerable economic and safety concerns, predominantly due to microbial corrosion. Early detection of corrosive microbes is pivotal for effective monitoring and prevention. Yet, traditional detection methods often lack specificity, require extensive processing time, and yield inaccurate results. Hence, the need for an efficient real-time corrosive microbe monitoring technology is evident. Pseudomonas aeruginosa, a widely distributed microorganism in aquatic environments, utilizes its production of quinone-like compounds, specifically pyocyanin (PYO), to corrode metals. Here, we report a novel fiber optic surface plasmon resonance (SPR) sensor modified by the C-terminal of BrlR protein (BrlR-C), which is a specific receptor of PYO molecule, to detect P. aeruginosa in aquatic environments. The results showed that the sensor had a good ability to recognize PYO in the concentration range of 0-1 µg/mL, and showed excellent sensing performance in real-time monitoring the growth status of P. aeruginosa. With a strong selectivity of PYO, the sensor could clearly detect P. aeruginosa against other bacteria in seawater environment, and exhibited excellent anti-interference ability against variations in pH, temperature and pressure and other interfering substances. This study provides a useful tool for monitoring corrosive P. aeruginosa biofilm in aquatic environments, which is a first of its kind example that serves as a laboratory model for the application of fiber optic technology in real-world scenarios to monitoring biofilms in microbial corrosion and biofouling.

Revealing roles of S-layer protein (SlpA) in Clostridioides difficile pathogenicity by generating the first slpA gene deletion mutant.

Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.

Surface roughness influence on extracellular electron microbiologically influenced corrosion of C1018 carbon steel by Desulfovibrio ferrophilus IS5 biofilm.

Carbon steel microbiologically influenced corrosion (MIC) by sulfate reducing bacteria (SRB) is known to occur via extracellular electron transfer (EET). A higher biofilm sessile cell count leads to more electrons being harvested for sulfate reduction by SRB in energy production. Metal surface roughness can impact the severity of MIC by SRB because of varied biofilm attachment. C1018 carbon steel coupons (1.2 cm2 top working surface) polished to 36 grit (4.06 µm roughness which is relatively rough) and 600 grit (0.13 µm) were incubated in enriched artificial seawater inoculated with highly corrosive Desulfovibrio ferrophilus IS5 at 28 â„ƒ for 7 d and 30 d. It was found that after 7 d of SRB incubation, 36 grit coupons had a 11% higher sessile cell count at (2.0 ± 0.17) × 108 cells/cm2, 52% higher weight loss at 22.4 ± 5.9 mg/cm2 (1.48 ± 0.39 mm/a uniform corrosion rate), and 18% higher maximum pit depth at 53 µm compared with 600 grit coupons. However, after 30 d, the differences diminished. Electrochemical tests with transient information supported the weight loss data trends. This work suggests that a rougher surface facilitates initial biofilm establishment but provides no long-term advantage for increased biofilm growth.

Conductive magnetic nanowires accelerated electron transfer between C1020 carbon steel and Desulfovibrio vulgaris biofilm.

Microbial biofilms are behind microbiologically influenced corrosion (MIC). Sessile cells in biofilms are many times more concentrated volumetrically than planktonic cells in the bulk fluids, thus providing locally high concentrations of chemicals. More importantly, "electroactive" sessile cells in biofilms are capable of utilizing extracellularly supplied electrons (e.g., from elemental Fe) for intracellular reduction of an oxidant such as sulfate in energy metabolism. MIC directly caused by anaerobic biofilms is classified into two main types based on their mechanisms: extracellular electron transfer MIC (EET-MIC) and metabolite MIC (M-MIC). Sulfate-reducing bacteria (SRB) are notorious for their corrosivity. They can cause EET-MIC in carbon steel, but they can also secrete biogenic H2S to corrode other metals such as Cu directly via M-MIC. This study investigated the use of conductive magnetic nanowires as electron mediators to accelerate and thus identify EET-MIC of C1020 by Desulfovibrio vulgaris. The presence of 40 ppm (w/w) nanowires in ATCC 1249 culture medium at 37 °C resulted in 45 % higher weight loss and 57 % deeper corrosion pits after 7-day incubation. Electrochemical tests using linear polarization resistance and potentiodynamic polarization supported the weight loss data trend. These findings suggest that conductive magnetic nanowires can be employed to identify EET-MIC. The use of insoluble 2 µm long nanowires proved that the extracellular section of the electron transfer process is a bottleneck in SRB MIC of carbon steel.

Mitigation of Desulfovibrio ferrophilus IS5 degradation of X80 carbon steel mechanical properties using a green biocide.

Most microbiologically influenced corrosion (MIC) studies focus on the threat of pinhole leaks caused by MIC pitting. However, microbes can also lead to structural failures. Tetrakis hydroxymethyl phosphonium sulfate (THPS) biocide mitigated the microbial degradation of mechanical properties of X80 steel pipeline by Desulfovibrio ferrophilus (IS5 strain), a very corrosive sulfate reducing bacterium. It was found that 100 ppm (w/w) THPS added to the enriched artificial seawater (EASW) culture medium before incubation resulted in 2.8-log reduction in sessile cell count after a 7-d incubation at 28 °C under anaerobic conditions, leading to 94% uniform corrosion rate reduction (from 1.3 to 0.07 mm/a), and 84% pitting corrosion rate reduction (from 0.70 to 0.11 mm/a). The X80 dogbone coupon incubated with 100 ppm THPS for 7 d suffered 3% loss in ultimate tensile strain and 0% loss in ultimate tensile strength compared with the abiotic control in EASW. In comparison, the no-treatment X80 dogbone coupon suffered losses of 13% in ultimate tensile strain and 6% in ultimate tensile stress, demonstrating very good THPS efficacy.

Electrochemical Assessment of Mitigation of Desulfovibrio ferrophilus IS5 Corrosion against N80 Carbon Steel and 26Cr3Mo Steel Using a Green Biocide Enhanced by a Nature-Mimicking Biofilm-Dispersing Peptide.

MIC (microbiologically influenced corrosion) is problematic in many industries, especially in the oil and gas industry. In this work, N80 carbon steel for pipelines was tested with 26Cr3Mo chromium pipeline steel for comparison in SRB (sulfate-reducing bacterium) MIC mitigation using a THPS (tetrakis hydroxymethyl phosphonium sulfate)-based commercial biocide (Biotreat 5475 with 75-80% THPS by mass). Peptide A, a nature-mimicking synthetic cyclic peptide (cys-ser-val-pro-tyr-asp-tyr-asn-trp-tyr-ser-asn-trp-cys) with biofilm dispersal ability was used as a biocide enhancer. Metal coupons covered with 3-d old Desulfovibrio ferrophilus IS5 biofilms were immersed in different biocide solutions. After 1-h treatment, 200 ppm Biotreat 5475, 200 ppm Biotreat 5475 + 200 nM (360 ppb) Peptide A, and 400 ppm Biotreat 5475 achieved 0.5-log, 1.7-log and 1.9-log reductions in sessile cell count on N80, and 0.7-log, 1.7-log, and 1.8-log on 26Cr3Mo, respectively. The addition of 200 nM Peptide A cut the THPS biocide dosage by nearly half. Biocide injection tests in electrochemical glass cells after 1 h exhibited 15%, 70%, and 72% corrosion inhibition efficiency (based on corrosion current density) on N80, and 27%, 79%, 75% on 26Cr3Mo, respectively. Linear polarization resistance and electrochemical impedance spectrometry results also indicated antimicrobial efficacies.

Mitigation of galvanized steel biocorrosion by Pseudomonas aeruginosa biofilm using a biocide enhanced by trehalase.

Pseudomonas aeruginosa is a facultative bacterium that is pathogenic. It is ubiquitous in the environment including air handling systems. It causes microbiologically influenced corrosion (MIC) aerobically and anaerobically. In this work, P. aeruginosa was grown as a nitrate reducing bacterium (NRB) in Luria-Bertani medium with KNO3 at 37 °C. Trehalase, an enzyme which plays a crucial role in biofilm formation was found to enhance the treatment of P. aeruginosa biofilm and its MIC against galvanized steel by tetrakis-hydroxymethyl phosphonium sulfate (THPS) green biocide. After a 7-d incubation, 30 ppm (w/w) trehalase reduced sessile cell count by 0.8-log, and it also reduced galvanized steel weight loss by 14%, compared to 2.3-log and 39%, respectively for the 30 ppm THPS treatment. The combination of 30 ppm THPS + 30 ppm trehalase reduced sessile cell count further by 0.1-log and weight loss by 13% compared to using THPS alone. Electrochemical corrosion measurements supported weight loss results. The injection of 20 ppm riboflavin into a 3-d P. aeruginosa broth failed to accelerate the corrosion rate, suggesting that nitrate reducing P. aeruginosa MIC of galvanized steel did not belong to extracellular electron transfer-MIC, because Zn was hydrolyzed after the microbe damaged the passive film.

Microbially mediated metal corrosion.

A wide diversity of microorganisms, typically growing as biofilms, has been implicated in corrosion, a multi-trillion dollar a year problem. Aerobic microorganisms establish conditions that promote metal corrosion, but most corrosion has been attributed to anaerobes. Microbially produced organic acids, sulfide and extracellular hydrogenases can accelerate metallic iron (Fe0) oxidation coupled to hydrogen (H2) production, as can respiratory anaerobes consuming H2 as an electron donor. Some bacteria and archaea directly accept electrons from Fe0 to support anaerobic respiration, often with c-type cytochromes as the apparent outer-surface electrical contact with the metal. Functional genetic studies are beginning to define corrosion mechanisms more rigorously. Omics studies are revealing which microorganisms are associated with corrosion, but new strategies for recovering corrosive microorganisms in culture are required to evaluate corrosive capabilities and mechanisms. Interdisciplinary studies of the interactions among microorganisms and between microorganisms and metals in corrosive biofilms show promise for developing new technologies to detect and prevent corrosion. In this Review, we explore the role of microorganisms in metal corrosion and discuss potential ways to mitigate it.

Effect of alloying element content on anaerobic microbiologically influenced corrosion sensitivity of stainless steels in enriched artificial seawater.

Stainless steels (SS) are not immune to microbiologically influenced corrosion (MIC) especially in the presence of sulfate reducing bacteria (SRB). It is necessary to study the influence of alloying elements on the MIC. SRB MIC behaviors of four stainless steels (2205 SS, 316L SS, 304 SS, and 410 SS), with different alloying element compositions were compared after 14 days of incubation at 37°C in enriched artificial seawater inoculated with Desulfovibrio sp. The sessile cell sequence was 410 SS > 316L SS > 304 SS > 2205 SS, inversely proportional to Cr content. The uniform corrosion rate (based on weight loss) sequence was 410 SS > 304 SS > 316L SS > 2205 SS, which matches the pitting resistance equivalent number (PREN) sequence inversely. 410 SS with the lowest Cr and Mo contents suffered the most severe pitting, with pit depth of 35 µm and weight loss of 0.75 mg/cm2 (0.91 mm/a pitting rate and 25 µm/a uniform corrosion rate). The other three stainless steels with higher Cr and Mo contents suffered only metastable pits. The semiconductor characteristics and the re-passivation abilities of the passive films were found to be affected by Cr and Mo contents.

Microbiologically influenced corrosion of titanium by Desulfovibrio vulgaris biofilm under organic carbon starvation.

Desulfovibrio vulgaris biofilm was pre-grown on Ti coupons for 7 d and then the biofilm covered coupons were incubated again with fresh culture media with 10 % (reduced) and 100 % (normal) carbon source levels, respectively. After the pre-growth, sessile D. vulgaris cell count reached 107 cells/cm2. The sessile cell counts were 2 × 107 and 4.2 × 107 cells/cm2 for 10 % and 100 % carbon sources, respectively after the subsequent 7 d starvation test. The maximum pit depth after the 7 d pre-growth was 4.7 µm. After the additional 7 d of the starvation test, the maximum pit depth increased to 5.1 µm for 100 % carbon source vs 6.2 µm for 10 % carbon source. Corrosion current density (icorr) from potentiodynamic polarization data at the end of the 7 d starvation test for 10 % carbon source was more than 3 times of that for 100 % carbon source, despite a reduced sessile cell count with 10 % carbon source. The polarization resistance (Rp) started to decrease within minutes after 20 ppm (w/w) riboflavin (electron mediator) injection. The carbon starvation data and riboflavin corrosion acceleration data both suggested that D. vulgaris utilized elemental Ti as an electron source to replace carbon source as the electron donor during carbon source starvation.

Multi-scale analysis of nickel ion tolerance mechanism for thermophilic Sulfobacillus thermosulfidooxidans in bioleaching.

Bioleaching is intensively investigated for recovering valuable metals such as Li, Co, Ni and Cu. Nickel ion stress threatens the health of microorganisms when Ni2+ starts to accumulate in the leachate during the bioleaching of materials that are rich in Ni, such as spent lithium-ion batteries. The possible mechanisms underlying the response of S. thermosulfidooxidans to nickel ion stress were analyzed using a multi-scale approach. Under the condition of nickel ion stress, high concentrations of nickel ions were immobilized by extracellular polymeric substances, while concentrations of nickel ions inside the cells remained low. The intracellular adenosine triphosphate (ATP) concentration and H+-ATPase activity increased to maintain normal cell growth and metabolic activities. Scavenging abilities of S. thermosulfidooxidans for hydrogen peroxide and superoxide anion were enhanced to reduce oxidative damage induced by nickel ion stress. There were 734 differentially expressed genes identified by RNA-seq under nickel ion stress. Most of them were involved in oxidative phosphorylation, glutathione metabolism and genetic information processing, responsible for intracellular energy utilization, intracellular antioxidant capacity and DNA damage repair, respectively. The results of this study are of major significance for in-depth understanding of the mechanisms of acidophilic microorganisms' resistance to metal ions.

Advances in bioleaching of waste lithium batteries under metal ion stress.

In modern societies, the accumulation of vast amounts of waste Li-ion batteries (WLIBs) is a grave concern. Bioleaching has great potential for the economic recovery of valuable metals from various electronic wastes. It has been successfully applied in mining on commercial scales. Bioleaching of WLIBs can not only recover valuable metals but also prevent environmental pollution. Many acidophilic microorganisms (APM) have been used in bioleaching of natural ores and urban mines. However, the activities of the growth and metabolism of APM are seriously inhibited by the high concentrations of heavy metal ions released by the bio-solubilization process, which slows down bioleaching over time. Only when the response mechanism of APM to harsh conditions is well understood, effective strategies to address this critical operational hurdle can be obtained. In this review, a multi-scale approach is used to summarize studies on the characteristics of bioleaching processes under metal ion stress. The response mechanisms of bacteria, including the mRNA expression levels of intracellular genes related to heavy metal ion resistance, are also reviewed. Alleviation of metal ion stress via addition of chemicals, such as spermine and glutathione is discussed. Monitoring using electrochemical characteristics of APM biofilms under metal ion stress is explored. In conclusion, effective engineering strategies can be proposed based on a deep understanding of the response mechanisms of APM to metal ion stress, which have been used to improve bioleaching efficiency effectively in lab tests. It is very important to engineer new bioleaching strains with high resistance to metal ions using gene editing and synthetic biotechnology in the near future.

Mechanical property degradation of X80 pipeline steel due to microbiologically influenced corrosion caused by Desulfovibrio vulgaris.

Apart from pinhole leaks, MIC (microbiologically influenced corrosion) can also cause catastrophic failures such as pipe ruptures and support beam collapses due to mechanical property degradation or stress corrosion cracking. In this work, X80 pipeline steel dogbone coupons and square coupons were immersed in 150 ml broths containing Desulfovibrio vulgaris, a common corrosive sulfate reducing bacterium (SRB), for up to 14 days. The headspace volumes in the anaerobic bottles were increased from 150 ml to 200 ml and 300 ml to increase MIC severity. After 14 days of SRB incubation in ATCC 1249 culture medium with X80 coupons at 37°C, the sessile cell counts were 6.5 × 107 cells cm-2 for 150 ml, 2.3 × 108 cells cm-2 for 200 ml and 1.4 × 109 cells cm-2 for 300 ml headspace volumes, respectively owing to reduced H2S cytotoxicity in the broth with a larger headspace because it allowed more biogenic H2S to escape from the broth. Weight losses were 1.7 mg cm-2, 1.9 mg cm-2 and 2.3 mg cm-2 for 150 ml, 200 ml and 300 ml headspace volumes, respectively. The corresponding pit depths were 2.6 µm, 4.2 µm and 6.2 µm for 150 ml, 200 ml and 300 ml headspace volumes, respectively. Electrochemical impedance spectroscopy (EIS), linear polarization resistance (LPR) and potentiodynamic polarization results corroborated the increasing weight loss and pitting data trends as a result of increased headspace. Tensile testing of dogbone coupons after the 14-day SRB immersion test indicated that more severe MIC pitting led to a higher ultimate strain loss by up to 23% (300 ml headspace) compared to the abiotic control, while the ultimate strength losses for all headspace volumes were quite small (3% and lower).

Preliminary Proof-of-Concept Testing of Novel Antimicrobial Heat-Conducting "Metallic" Coatings Against Biofouling and Biocorrosion.

NiMo (nickel-molybdenum) and NiMo with embedded CeO2 nanoparticles (NPs; 100 nm) were tested as antimicrobial coatings (~15 µm thickness) on titanium (Ti) surfaces using an electrochemical process for heat exchanger applications onboard marine vessels. Preliminary static biofouling and biocorrosion (also known as microbiologically influenced corrosion) assessments were carried out in glass bottles using pure-culture Desulfovibrio vulgaris, a sulfate-reducing bacterium (SRB), in deoxygenated ATCC 1249 medium at 37°C, and using an alga (Chlorella vulgaris) mixed with general heterotrophic bacteria (GHB) in enriched artificial seawater at 28°C. It was found that the coating containing NiMo/CeO2 NPs were much more effective than NiMo in preventing SRB biofilm formation with an efficacy of 99% reduction in D. vulgaris sessile cells after 21 day incubation. The coating also exhibited a 50% lower corrosion current density compared to the uncoated Ti against SRB corrosion. Both NiMo and NiMo/CeO2 NP coatings achieved 99% reduction in sessile algal cells. Confocal laser scanning microscopy (CLSM) biofilm images indicated a large reduction of sessile GHB cells. The CLSM images also confirmed the biocidal kill effects of the two coatings. Unlike polymer coatings, the "metallic" coatings are heat conductive. Thus, the corrosion resistant antifouling coatings are suitable for heat exchanger applications.

Strategies for anti-oxidative stress and anti-acid stress in bioleaching of LiCoO2 using an acidophilic microbial consortium.

High metal ion concentrations and low pH cause severely inhibit the activity of an acidophilic microbial consortium (AMC) in bioleaching. This work investigated the effects of exogenous spermine on biofilm formation and the bioleaching efficiency of LiCoO2 by AMC in 9K medium. After the addition of 1 mM spermine, the activities of glutathione peroxidase and catalase increased, while the amount of H2O2, intracellular reactive oxygen species (ROS) and malondialdehyde in AMC decreased. These results indicated that the ability of AMC biofilm to resist oxidative stress introduced by 3.5 g/L Li+ and 30.1 g/L Co2+ was improved by spermine. The activity of glutamate decarboxylase was promoted to restore the intracellular pH buffering ability of AMC. Electrochemical measurements showed that the oxidation rate of pyrite was increased by exogenous spermine. As a result, high bioleaching efficiencies of 97.1% for Li+ and 96.1% for Co2+ from a 5.0% (w v-1) lithium cobalt oxide powder slurry were achieved. This work demonstrated that Tafel polarization can be used to monitor the AMC biofilm's ability of uptaking electrons from pyrite during bioleaching. The corrosion current density increased with 1 mM spermine, indicating enhanced electron uptake by the biofilm from pyrite.

Direct microbial electron uptake as a mechanism for stainless steel corrosion in aerobic environments.

Shewanella oneidensis MR-1 is an attractive model microbe for elucidating the biofilm-metal interactions that contribute to the billions of dollars in corrosion damage to industrial applications each year. Multiple mechanisms for S. oneidensis-enhanced corrosion have been proposed, but none of these mechanisms have previously been rigorously investigated with methods that rule out alternative routes for electron transfer. We found that S. oneidensis grown under aerobic conditions formed thick biofilms (∼50 µm) on stainless steel coupons, accelerating corrosion over sterile controls. H2 and flavins were ruled out as intermediary electron carriers because stainless steel did not reduce riboflavin and previous studies have demonstrated stainless does not generate H2. Strain ∆mtrCBA, in which the genes for the most abundant porin-cytochrome conduit in S. oneidensis were deleted, corroded stainless steel substantially less than wild-type in aerobic cultures. Wild-type biofilms readily reduced nitrate with stainless steel as the sole electron donor under anaerobic conditions, but strain ∆mtrCBA did not. These results demonstrate that S. oneidensis can directly consume electrons from iron-containing metals and illustrate how direct metal-to-microbe electron transfer can be an important route for corrosion, even in aerobic environments.

Bacterial biofilms as platforms engineered for diverse applications.

Historically, biofilms have been perceived as problematic or detrimental. However, biofilms possess favorable traits such as self-regeneration, sustainability, scalability, and tunability, which make them candidates for diverse applications. Traditional applications of biofilms, such as environmental remediation, bioleaching, microbial fuel cells, and corrosion protection, are often built on the basis of wild-type or metabolically engineered strains. In this review, we further comment on the design strategies for multiple innovative applications of living functional biofilms. With the integration of signaling pathways, engineering of metabolic pathways and modification of extracellular polymeric substances, living functional biofilms have been constructed by researchers through various strategies. Functional biofilms for diverse applications, including catalysis, electric conduction, bioremediation, and medical therapy have been demonstrated in the literature. The mechanical properties of biofilms can be tuned through genetic editing, metal ion curing and synthetic gene circuits, etc. In addition, the improvement of 3D printing to use bioinks has also achieved significant progresses in fabricating living functional biofilms with specific structures. In the future, the combination of synthetic biology and techniques from other disciplines will lead to practical large-scale applications of biofilms.