Methods and apparatus to monitor biofilms, biocorrosion, and antimicrobial biofilm treatment

Abstract

Apparatus, systems and methods for the detection of biofilms and biocorrosion, distinguishing different types of biocorrosion, and the assessment of antimicrobial treatment of biofilms and biocorrosion offline and online. An embodiment of an apparatus includes an electrochemical cell including first and second solid-state electrodes with the first solid-state electrode being a working electrode and the second solid-state electrode being a counter electrode. The counter electrode also functions as a pseudo-reference electrode.

Description

METHODS AND APPARATUS TO MONITOR BIOFILMS, BIOCORROSION, AND ANTIMICROBIAL BIOFILM TREATMENT

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to, and the benefit of the tiling date of, U.S. Provisional Application No. 63 / 510,940, filed on June 29, 2023, U.S. Provisional Application No. 63 / 622,849, filed on January 19, 2024, and U.S. Provisional Application No. 63 / 561,883, filed on March 6, 2024, the disclosures of which are incorporated herein by reference in their entireties.

[0003] FIELD OF THE INVENTION

[0004] The present invention generally relates to (1) the detection of biofilms and biocorrosion, (2) distinguishing different types of biocorrosion, and (3) the assessment of antimicrobial treatment of biofilms and biocorrosion offline and online.

[0005] BACKGROUND OF THE INVENTION

[0006] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior ail.

[0007] Microbial biofilms contain sessile microbes embedded in exopolymeric substances (EPS). Most microbes in nature live in biofilm communities [1], Biofilms are synergistic communities, allowing microbes to share resources and offering protections against harmful environmental conditions such as attacks by antimicrobials [2,3]. It is known that sessile cells in biofilms are far more recalcitrant than their planktonic counterparts. A much higher antimicrobial dosage is needed to treat sessile cells than treating planktonic cells.

[0008] Some biofilms are beneficial. For example, environmental biofilms in wetlands help clean up water pollution [4,5], However, problematic biofilms cause various problems such asbiofouling [6], microbiologically influenced corrosion (MIC) [7], and diseases [8], In MIC, sulfate reducing bacteria (SRB) are notoriously corrosive.

[0009] Part of the reason as to why microbes in a biofilm can be more problematic than planktonic is because biofilms offer an ecological environment that can be very different from the bulk-fluid phase. Biofilms have a sessile cell density easily 100X or 1000X higher per unit volume than planktonic cells in the bulk-fluid phase. This means, underneath a biofilm, the biochemical conditions can be vastly different from those in the bulk-fluid phase. Thus, harmful metabolites can have much higher concentrations underneath a biofilm. For example, the pH (indicator of H+concentration) underneath an acid producing bacterium (APB) biofilm can be far more acidic, which can lead to acid corrosion of metals. Unlike planktonic cells, some sessile cells can harvest electrons from a metal via extracellular electron transfer (EET), causing EET-MIC (Li et al., 2018). For pathogenic microbes, a biofilm can be far more virulent than suspended planktonic cells simply because of a locally much higher cell concentration underneath a biofilm.

[0010] MIC is a major problem in many industries including oil and gas, water utilities, and power generation. Corrosion can be problematic for the interior as well as exterior of pipelines and other assets, and results in an annual cost of US$4 trillion globally

[0047] . MIC is responsible for 20% of all corrosion damages [48,49]. It happens in various industrial environments such as underground pipelines, sewage water conduits, single-pass and recirculating cooling systems, and oil and gas wells [50-54], Many microbes can cause MIC. Among them, sulfate reducing bacteria (SRB) - such as Desulfovibrio ferrophilus (strain IS5) - and acid producing bacteria (APB) are the most notorious

[0038] , leading to large economic losses [9]. The majority of oil and gas MIC cases involve SRB because seawater flooding is often used to increase reservoir pressure and some seawater later gets into pipelines. MIC pitting rates as high as 1.5 cm / year have been demonstrated in lab testing

[0039] . Biofilms inside an oil and gas pipeline are not routinely sampled by operators due to operational difficulties. They may come from pig trap sludge after a pigging run. However, many pipelines cannot be pigged due to their complex structures. Planktonic cell counts are often monitored instead, but they do not necessarily reflect sessile cell composition and concentration. As a compromise, biofilms grown on carbon steel (chosen to be the same metal as the pipeline steel) using the pipeline fluid is the next best thing. An online biofilm / MIC sensor can also be used, but that requires drilling a hole in thepipeline or a storage tank. With a test kit or an online sensor, corrosion rate can be measured from electrochemical scan data, and the growth of corrosive biofilms and biocide treatment efficacies can be assessed based on corrosion rate change.

[0011] To monitor biofilm growth / health and antimicrobial biofilm treatment efficacy, biofilms on a solid surface can be imaged directly using confocal laser scanning microscopy (CLSM) with stains that identify live and dead cells

[0010] . Proteins, polysaccharides and nucleic acids in a biofilm can also be used to quantify the biofilm’s biomass, but they do not necessarily correlate with sessile cell count since EPS components include extracellular proteins, polysaccharides and nucleic acids [11,12], Microbiology methods can be used to quantify sessile cells in biofilms. Sessile cells can be scraped off a solid surface first, and then resuspended in a liquid for analysis of cell types and cell quantities. For example, a hemocytometer can be used to count cell particles in a cell suspension if they are large enough to be seen or they are motile, which makes them viewable under a microscope at 400X magnification. Various microbiology test kits can be used to identify and count sessile cells. For example, there are test kits to grow SRB cells and acid producing bacteria cells first and then quantifying them [13,14]. When re-suspended sessile cells are very dilute in a cell suspension (e.g., after a biocide treatment), the most probable number (MPN) method is often used relying on exhaustive serial dilutions, aiming at the last test tube not having a single cell so that the test tube does not show growth (e.g., indicated by turbidity or color in the culture medium) while the test tube before that shows growth after 14, 21, or as long as 28 days. Optical density can also be used to quantify cells in a cell suspension if the culture medium background color or precipitates interference can be eliminated.

[0012] However, a drawback is that none of these methods can be used to monitor biofilm growth in situ continuously. Although some imaging methods like CLSM can visualize biofilms while they are on their original surfaces, they change the biofilm growth environment with stains, and anaerobic biofilms will be exposed to O2 during imaging. Other methods require scraping off sessile cells to re-suspend them.

[0013] Although there are some commercial test kits claiming to be MIC test kits, they are actually microbiology test kits, because they don’t measure corrosivity at all [15,16]. It wouldbe desirable to develop a simple to use an inexpensive / disposable apparatus that can be used to assess biofilm growth and biofilm treatment continuously.

[0014] MIC researchers have been using various electrochemical methods to measure biofilm corrosivity and corrosivity reduction after biofilm treatment in lab tests. For the same microbe, a biofilm with more sessile cells is more corrosive. For example, in extracellular electron transfer (EET) MIC (EET-MIC), more sessile cells harvest more electrons as in the case of SRB MIC of carbon steel

[0017] , For metabolite-MIC (M-MIC), such as SRB MIC of copper due to corrosive biogenic FhS, a biofilm with more sessile cells secretes more corrosive metabolites underneath it.

[0015] One commonly used electrochemical measurement in corrosion is open circuit potential (OCP). The electrochemical scan is performed using a potentiostat (also known as an electrochemical workstation). OCP is a passive measurement method without using an externally imposed electric field. In the inventors’ research, the inventors discovered that passive measurements such as OCP and microbial fuel cell output measurements for MIC are often unreliable and misleading due the complexity of the corrosion system. Electrochemical methods can be extremely sensitive. For example, titanium corrosion is extremely slow and very difficult to measure in terms of weight loss and corrosion pit depth. However, its electrochemical corrosion measurements can easily demonstrate MIC rates that are affected by biofilm and biofilm mitigation

[0018] . Thus, to exhibit measurable corrosivity electrochemically, a biofilm does not need to produce microscopically visible corrosion in terms of measurable weight loss and pit depth. This means many biofilms that are not known to be corrosive (due to their very low corrosivity) can be electrochemically monitored for their corrosivity change, which will reflect the corresponding biofilm growth / health.

[0016] However, in MIC, OCP is often misleading because a lower OCP theoretically merely means a higher tendency for corrosion rather than corrosion rate. A higher tendency for coiTosion can be offset by slow kinetics. The higher tendency for corrosion may come from a corrosion process that is not responsible for the actual corrosion because it is kinetically too retarded. Instead, a different corrosion process may be responsible, but its OCP may be much higher (i.e., lower tendency for corrosion). Thus, it is not reliable to use OCP to predict biocorrosion.

[0017] In addition to internal MIC commonly seen in pipelines, external MIC is observed as well [55,56], MIC was also found to be the predominant factor responsible for the perforation and leakage of buried pipelines in soil environments, which arc typical examples of external MIC [57,58],

[0018] MIC of metals is the result of interactions between metals, surrounding environments and microorganisms

[0059] , During the soil MIC process, external corrosion of buried pipelines happens when corrosive microorganisms facilitate the electron transport from metals to suitable electron acceptors such as H+, sulfate and nitrate [60,61], Sulfate reducing bacteria (SRB) play an important role in soil MIC of pipelines since the oxygen level in soil is usually low, which promotes SRB growth

[0062] , They are very aggressive to pipelines as they can directly or indirectly utilize Fe° as electron donors to harvest energy under the condition of organic carbon starvation [63,64], Apart from SRB, nitrate reducing bacteria (NRB) is another important group that live in soil. They were also reported to be responsible for severe MIC under anaerobic conditions [65,66], The wide application of nitrogen fertilizer and biological nitrogen fixation in soil provide NRB a suitable environment to grow [61,67,68], NRB are also capable of causing MIC when organic carbon source is insufficient

[0069] .

[0019] The use of a coating is considered an effective approach to prevent corrosion, as a coating offers a physical barrier between the metallic surface of the pipeline and the aggressive environment

[0070] , Different materials such as chromate-based conversion coatings

[0071] , anodized coatings

[0072] , metallic and organic coatings are widely used to combat corrosion

[0073] . Due to the low cost, versatility and aesthetic attributes, organic coatings have become the single most widely applied method in corrosion protection [74,75], Besides the barrier effect, organic coatings can also have protective mechanisms which offer inhibitive effect, anodic protection and cathodic protection

[0076] , Common organic coatings include organic conversion coatings, thermoplastics, thermosets (e.g., Epoxy resins), elastomers and organic zinc-rich coatings

[0077] ,

[0020] However, there are still several factors that limit the application of organic coatings. Some solvent based organic coatings pose a significant environmental issue

[0078] , Besides, multiplied layers of organic coatings usually need to be applied to ensure sufficient corrosionresistance since it is difficult to create a uniform layer without pores

[0079] . Most importantly, the adhesion of the organic coatings to substrate might be poor without appropriate pretreatment

[0080] , The adhesion can also be reduced by water molecules at the interface between metals and organic coatings

[0081] . Coating disbondment is another issue that may lead to coating failures. Blistering, anodic undermining and cathodic disbondment are main causes of coating disbondment

[0082] , When coating failures happen, corrosive species get in contact with metal substrate and will promote corrosion. Therefore, it is important to detect coating failures promptly to stop corrosion development. However, currently there are no efficient and effective ways to accomplish this.

[0021] Apart from the issues that microbes cause with respect to biocorrosion (such as internal and external to pipelines, as described above), microbial biofilms are also involved in diseases. For example, Pseudomonas aeruginosa biofilm is involved in cystic fibrosis (Hassett et al., 2010). Clostridioides difficile biofilm causes diarrhea and colitis (food-bone diseases) according to US CDC (https : / / www.cdc. gov / ediff / what-is .htrnl) . Shigella flexneri biofilm is known to cause colonic infection (Chiang et al., 2021). Biofilms can grow on the internal walls of catheters, such as those used for chemotherapy patients, leading to lifethreatening infections because these patients have weakened immune system during chemotherapy (Raad et al., 2007).

[0022] Biofilms are notoriously recalcitrant toward antimicrobial treatment (Donlan, 2008; Gominet et al., 2017), often requiring 10X higher dosage of antimicrobials (Hall-Stoodley et al., 2004), because they have various defense mechanisms (Jia et al., 2019). For example, Pseudomonas aeruginosa infects the air way of cystic fibrosis patients and it is very difficult to treat (Hassett et al., 2010). The prevalence of biofilms in chronic wounds was found to be 78.2 % (Malone et al., 2017).

[0023] To enumerate sessile cells in biofilms, sessile cells are scraped off a solid surface and re-suspended in water or a buffer. Motile bacterial cells can be counted rather accurately using a hemocytometer on a light microscope at 400X magnification because the very small cell dots can be differentiated from artifacts. In antimicrobial treatment studies, a cell suspension’s cell count may be well below the 50,000 cells / mL threshold for accurate hemocytometer counting (of motile bacterial cells) or other imaging methods with stains suchas CLSM (confocal laser scanning microscopy) under 400X microscope, because cells are rarely seen in the view field (i.e., not enough of them to be seen). Using 40X magnification can cover a 100X larger view area than 400X magnification, but the bacterial cells are too small to be identifiable even for motile cells or stained cells. MPN (most probably number) serial dilution method can be used to grow cells for counting. CFU (colony forming unit) in the plating method can also be used to grow colonies for counting. These methods are timeconsuming and not very accurate. Cell counts / concentrations in medical biofilm studies often carry wide error bars, making statistical significance hard to achieve in many cases.

[0024] Because antibiotic resistance is a serious concern nowadays, there is need to accelerate the antibiotic efficacy assessment in vivo and to accelerate the development of new antibiotics or the use of combinations of existing antibiotics. Thus, a high-throughput and convenient way to assess antimicrobial efficacies is highly desirable.

[0025] SUMMARY OF THE INVENTION

[0026] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0027] As noted above, there are several drawbacks to current methods and apparatus for detecting biofilms and biocorrosion, for distinguishing different types of corrosion (such as biotic versus abiotic), and for assessing antimicrobial treatment of biofilms and biocorrosion offline and online.

[0028] Currently expensive and large sensors are used. These sensors cannot distinguish biocorrosion type. They cannot distinguish abiotic corrosion from biocorrosion without using an antimicrobial.

[0029] Various aspects of the present invention reduce these drawbacks by providing a test kit that is small in low cost and disposable. It can detect corrosion rate and corrosion type. It canbe widely used to provide numerous data on biocorrosion. Currently, microbiology test kits only provide microbe information rather than biocorrosion information.

[0030] One aspect of the present invention accomplishes this by providing an apparatus including an electrochemical cell including first and second solid-state electrodes. In this embodiment, the first solid-state electrode is a working electrode, and the second solid-state electrode is a counter electrode. In particular, the electrochemical cell of this aspect of the present invention does not include a third electrode - being a reference electrode - in addition to the first and second solid-state electrodes. Rather, the counter electrode also functions as a pseudo-reference electrode.

[0031] In another embodiment, a third dedicated reference electrode that is a solid-state electrode can be used rather than using a pseudo -reference electrode.

[0032] A user can introduce a fluid or liquid suspension of particles (e.g., sludge) for incubation to grow biofilms on the working electrode of the apparatus. Various electrochemical scans may then be performed to distinguish abiotic corrosion from biocorrosion, to detect biocorrosion rate, and to assess biocide treatment efficacy.

[0033] In one example of a particular embodiment, the apparatus may be a miniature electrochemical cell. For example, the cell may include as small as a 10 mL serum vial with two solid-state electrodes and a rubber septum. A 1 cm2metal coupon may be used as working electrode. And a 1 cm2graphite or other conductive material may serve as the counter electrode / pseudo-reference electrode combination. As described above, a user can inject a fluid or liquid suspension of particles for incubation to grow biofilms on the working electrode. Various electrochemical scans may then be performed periodically to distinguish abiotic corrosion from biocorrosion, and to detect biocorrosion rate during an incubation period that lasts days.

[0034] As described above, one commonly used electrochemical measurement in corrosion is open circuit potential (OCP), but the inventors discovered that passive measurements such as OCP and microbial fuel cell output measurements for MIC are often unreliable and misleading due the complexity of the corrosion system. As noted, OCP is often misleading because alower OCP theoretically merely means a higher tendency for corrosion rather than corrosion rate. A higher tendency for corrosion can be offset by slow kinetics. The higher tendency for corrosion may come from a corrosion process that is not responsible for the actual corrosion because it is kinetically too retarded. Instead, a different corrosion process may be responsible, but its OCP may be much higher (i.e., lower tendency for corrosion). Thus, while one may use OCP to predict corrosion, its use may not be as accurate as some other methods.

[0035] In that regard, the inventors have discovered that non-passive linear polarization resistance (LPR), Tafel (also known as potentiodynamic polarization), electrochemical impedance spectroscopy (EIS), and electrochemical frequency modulation (EFM) scans are reliable in predicting corrosion trends. Electrochemical scans are performed using a potentiostat (also known as an electrochemical workstation). OCP is a passive measurement method without using an externally imposed electric field. LPR and Tafel scans use an externally imposed DC (direct current) electric field for scanning, while EIS and EFM use AC (alternate current) electric fields. Thus, the inventors have determined that kinetic scans (such as LPR, Tafel, EIS and EFM) can be relied upon to give more accurate measurements.

[0036] As noted above, such an apparatus can be used as an online sensor device with two solid-state electrodes to detect biocorrosion in the area of pipelines (although the use of the apparatus would not be limited to pipelines (such as oil or gas pipelines) - but could be applicable to any conduit and any storage vessel, or indeed to any component that houses or transports a fluid - or is simply subject to corrosion.

[0037] Thus, another aspect of the present invention is directed to a system for the detection of biofilms and biocorrosion, for the assessment of biofilms and biocorrosion, and for the assessment of treatment of same, the system comprising a component that is potentially subject to biocorrosion or to biofilms associating therewith, and an apparatus (including electrochemical cell) as described above.

[0038] In one aspect, the apparatus may be a stand-alone apparatus (i.e., offline or separate from the component, e.g., pipeline or other conduit). The material of the working electrode of the apparatus may be of the same material that forms the component (e.g., pipeline or other conduit). A sample of fluid from the pipeline can then be introduced into the electrochemicalcell (such as via injection through a septum that closes the housing of the electrochemical cell. Various electrochemical scans may then be performed to distinguish abiotic corrosion from biocorrosion, and to detect biocorrosion rate.

[0039] In another aspect, the apparatus may be associated with the component (e.g., pipeline or other conduit), such as by being affixed thereto and allowing for direct introduction of electrodes to fluid in the component, or introduction of fluid into electrochemical cell. For example, part of the housing of the electrochemical cell may be contiguous with a wall of the pipeline. There may be a valve between the pipeline and cell. And the valve may allow for the first and second electrodes of the electrochemical cell to pass through the valve into the interior space of the pipeline to contact fluid therein. The electrodes may then be retracted through the valve and back into the electrochemical cell and becomes isolated from the bulk pipeline fluid when the valve is closed, which allows chemical injections into the isolated electrochemical cell for corrosion type diagnosis and biocide testing. Additionally, or alternatively, the valve may allow for fluid from the pipeline to pass into the cell when the valve is opened so the microbes in the fluid can build a biofilm on the working electrode of the electrochemical cell.

[0040] In addition to detecting, assessing, and assessing treatment of biofilms and corrosion (such as biocorrosion) interior to a component (such as a pipeline), another aspect of the present invention is directed to detecting, assessing, and assessing treatment of biofilms and corrosion (such as biocorrosion) on the external surface of a component (such as a pipeline), or to a coating on such external surface.

[0041] As noted previously, coatings are used to protect various metal surfaces against corrosion / biocorrosion. They can be damaged by scratches and disbondment. Corrosive chemicals and microbes can access the metal and cause corrosion / biocorrosion. Thus, as noted above, there is a need to monitor damages to coatings on various assets such as industrial, marine, and military assets.

[0042] In this aspect of the present invention, an apparatus may be used similar to that described above with respect to testing the interior of various components. In that regard, then, this aspect of the present invention may include providing an apparatus including anelectrochemical cell including first and second solid-state electrodes. In this embodiment, the first solid-state electrode is a working electrode, and the second solid-state electrode is a counter electrode. In particular, the electrochemical cell of this aspect of the present invention docs not include a third electrode - being a reference electrode - in addition to the first and second solid-state electrodes. Rather, the counter electrode can also function as a pseudoreference electrode.

[0043] In this aspect, the present inventors adopt solid-state electrodes (as disclosed above) to monitor corrosion resistance and corrosion rate using electrochemical methods including linear polarization, potentiodynamic polarization and electrochemical impedance spectroscopy scans. The working electrode (WE) can be the base metal under the coatings or a more easily corroded metal covered with (to be damaged) coatings. The counter electrode / pseudo-reference electrode (CE / p-RE) (combination electrode) can be graphite, a conductive polymer, stainless steel and other metals (e.g., metal containing platinum, cerium, chromium, nickel). The electrodes have a small working surface (as small as 1 cm2or less, though they can be slightly larger). The WE and CE / p-RE are solid-state materials. The two electrodes are connected to a potentiostat to perform the electrochemical scans periodically. If corrosion rate becomes significant (or corrosion resistance drops significantly), it means the coatings are damaged.

[0044] In certain embodiments of the apparatus that are used both internally and externally to a component (such as a pipeline) only solid-state electrodes are used. Because of the use of solid-state electrodes, no electrolyte-filled reference electrode is needed. Additionally, the electrodes that are used can be quite inexpensive (for example, even Pt coated electrodes are fairly inexpensive). These inexpensive solid-state electrodes can be used in numerous places on an asset such as a pipeline's external surface which is prone to soil or sludge or polluted water corrosion (with and without microbes to contribute to the corrosion) once coatings are damaged.

[0045] Due to its small size, the sensor can be portable / mobile to detect damages in coatings in different places, or an online sensor can be deployed at a fixed location. Additionally, due to the determination by the inventors that even inexpensive electrodes can work effectively (and in such a small apparatus), the apparatus is not only portable, but can also be disposable (though it can also be reused if cleaned and sanitized).

[0046] Due to the nature, materials, size, etc. of the apparatus of the present invention, the apparatus provides fast scans (e.g., as fast as 2 minutes per scan) and are able to provide transient corrosion data to indicate damages in coatings that lead to corrosion. Further, the apparatus is capable of identifying disbondment (indicated by a large corrosion rate increase detected by the sensor) in addition to scratches. The apparatus can be easily embedded on buried pipelines. It can be used when submerged in field liquid such as rain water or sewer water. And it can be used to distinguish abiotic corrosion from biocorrosion after coatings are damaged (as will be described in greater detail below).

[0047] Thus, as described above, the present inventors have determined that kinetic scans (LPR, Tafel, EIS and EFM) are more reliable than (passive) OCP. And so one may desire to use one or more of these kinetic scans. Among these electrochemical methods, LPR is the fastest. Each scan normally takes 2 min to complete, and it can be reduced to 1 min if needed. Tafel, EIS and EFM scans take about 10 times longer, as described above). Among these methods, EIS is particularly useful in testing integrity of coatings

[0020] .

[0048] Another aspect of the present invention is directed to the use of the apparatus described herein to test the antimicrobial efficacy of a medical biofilm. It can be used in assessing the growth of environmental biofilms that cause biofouling and biocorrosion, as well as assessing the efficacy of various treatments against these biofilms.

[0049] As noted above, current methods and apparatus for assessing antibiotics efficacy against medical biofilms that cause diseases are tedious and the efficacy data generated tend to have large error bars. The use of the apparatus of the present invention is relatively simple, and can provide transient information on efficacy that is difficult to obtain using existing methods. Further, the data generated in the apparatus, systems, and methods of the present invention, result in less uncertainties in judging efficacy.

[0050] As discussed above in the Background section, antimicrobial efficacy testing usually is done by enumerating sessile cells in biofilms with and without antimicrobial treatment. To enumerate sessile cells in biofilms, sessile cells are scraped off a solid surface and re-suspended in water or a buffer. Motile bacterial cells can be counted rather accurately using ahemocytometer at 400X magnification because the very small cell dots can be differentiated from artifacts. In antimicrobial treatment studies, a cell suspension’s cell count may be well below the 50,000 cells / mL threshold for accurate hemocytometer counting (of motile bacterial cells) or other imaging methods with stains such as CLSM (confocal laser scanning microscopy) under 400X microscope, because cells are rarely seen in the view field (i.e., there arc not enough of them to be seen). Using 40X magnification can cover a 100X larger view area than 400X magnification, but the bacterial cells are too small to be identifiable even for motile cells or stained cells. The Most Probable Number (MPN) serial dilution method can be used to grow cells for counting. The Colony Forming Unit (CFU) plating method can also be used to grow colonies for counting. However, as is well known by those of ordinary skill in the ail, these methods are time-consuming and not accurate. Cell counts in medical biofilm studies often carry wide error bars, making statistical significance hard to achieve in many cases.

[0051] The apparatus in various aspects of the present invention, however, makes testing easy. A user only needs to introduce sample into the apparatus of the present invention (e.g., by making injections into the electrochemical cell, i.e., electrochemical test biofilm / MIC test kit, described herein), and then perform electrochemical scans. The methods described herein provide more data [such as minimum dosage, time to kill, kill persistence (time for biofilm to recover), prevention persistence, and efficacy level (reflected by corrosion rate reduction)]. Additionally, multiple apparatus can be run at the same time for assessing multiple samples at the same time to achieve high throughput.

[0052] Another aspect of the invention may include a method for detecting biofilms and biocorrosion, for distinguishing types of corrosion, or for assessing antimicrobial treatment of biofilms and biocorrosion. The method may include the steps of (1) obtaining a sample from a component (such as those described above) having at least one surface subject to corrosion; (2) introducing the sample to an apparatus including an electrochemical cell comprising first and second solid-state electrodes, wherein the first solid-state electrode is a working electrode and the second solid-state electrode is a counter electrode (wherein the electrochemical cell does not include a dedicated reference electrode in addition to the first and second solid-state electrodes); and (3) testing the working electrode to determine if a biofilm has grown on the working electrode.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The accompanying drawings, which arc incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the general description of the invention given above and the detailed description of the embodiments given below, serve to explain the principles of the present invention.

[0055] FIG. 1 are photographs of a 10 mL anaerobic vial (serum vial) converted to a miniature electrochemical glass cell (before use and after 7-day SRB incubation) with black glue on top to seal better) serving as a biofilm / MIC test kit to measure biofilm growth, MIC rate and to distinguish abiotic corrosion from MIC, and EET-MIC from M-MIC. The glass cell has a carbon steel electrode to match that of a pipeline steel and a solid-state graphite electrode serving as CE and p-RE at the same time.

[0056] FIG. 2 are photographs showing biofilm / MIC test kit examples with larger vial sizes (20 mL and 50 mL).

[0057] FIGS. 3A-D are drawings [in FIG. 3A and 3D] and photographs [in FIGS. 3B and 3C] showing: (1) FIG. 3 A is a classical 3-electrode (3E) setup for electrochemical glass cell. For 2-electrode (2E) electrochemical testing, the non- solid- state saturated calomel electrode (SCE) reference electrode (RE) is not used. A platinum mesh serves as both counter electrode and pseudo-RE at the same time. (2) FIG. 3B is a photograph of a 450 mL 3E cell after and before SRB incubation. (The large size and a non- solid- state dedicated RE make the 3E device not suitable for field uses, but nonetheless useful in validating 2E scans in small electrochemical cells.). (3) FIG. 3C is an image of a SCE. FIG. 3D is a schematic of a 2- electrode (2E) electrochemical testing, where the non- solid- state SCE reference electrode is not used; rather the counter electrode serves as a pseudo-reference electrode (e.g., a platinum mesh serves as both counter electrode and pseudo-RE at the same time).

[0058] FIG. 4 is a graph showing LPR scans with and without RE (3-electrode system vs. 2- electrode system) both showing increased MIC of C1018 carbon steel after electron mediator (riboflavin) injection into 3-d nitrate-reducing P. aeruginosa broth in a 450 mL glass cellwith 250 mL Luria-Bertani medium with KNO3 (LB-NO3) culture medium at 37°C, proving that a dedicated RE is not essential in providing corrosion trends.

[0059] FIG. 5 is a graph showing variations of X65 WE Rpduring 7-d incubation of D. ferrophilus in 5 mL EASW in 10 mL biofilm test kit at 28°C. (Range bar obtained from two replicate 10 mL glass cells.)

[0060] FIG. 6 is a graph showing corrosion current density (Lorr) values fitted from Tafel scans during 7-d incubation of D . ferrophilus in 5 mL EASW in 10 mL biofilm test kit.

[0061] FIG. 7 is a graph showing corrosion rate trend based on EFM results during 7-d incubation of D . ferrophilus in 5 mL EASW in 10 mL biofilm test kit.

[0062] FIG. 8 is a graph showing variations of (Rf + Rct) of X65 WE fitted from EIS data during 7-d incubation of D. ferrophilus in 5 mL EASW in 10 mL biofilm test kit, in which Rf (resistance of the film consisting of biofilm and corrosion products) and Rct (charge transfer resistance) data were from EIS spectrum modeling using an equivalent circuit.

[0063] FIG. 9 is a graph showing LPR scan showing increased corrosion after electron mediator (riboflavin) injection (into 3-d old broth), and decreased corrosion after THPS biocide injection for SRB MIC of X65 pipeline steel by D. ferrophilus (a very corrosive SRB strain) in a 10 mL biofilm test kit containing 7 mL EASW at 28°C.

[0064] FIG. 10 is a graph showing corrosion current density (jcorr) time profile from Tafel scans on several days for MIC of X65 pipeline steel by D. ferrophilus SRB in a 10 mL miniature 2E electrochemical cell (i.e., Ohio University biofilm / MIC test kit) containing 7 mL EASW.

[0065] FIG. 11 is a graph showing LPR scan profile of 10 mL test kit containing 7 mL 5% (w / w) NaCl solution pre-sparged with CO2 (pH 4) abiotically corroding X65 carbon steel WE with a graphite electrode serving as both CE and pseudo-RE, showing injection disturbance, but not increased corrosion for riboflavin (RF) electron mediator injection or decreased corrosion after THPS biocide injection, thus suggesting abiotic corrosion.

[0066] FIG. 12 is a graph showing LPR scan profile of 10 mL test kit containing 7 mL 5% (w / w) NaCl solution pre-sparged with CO2 (pH 4) abiotically corroding X65 carbon steel WE with a graphite electrode serving as CE / p-RE, showing injection disturbance, but no increased corrosion for riboflavin (RF) electron mediator injection or decreased corrosion after THPS biocide injection, thus indicating abiotic corrosion.

[0067] FIG. 13 is a graph showing Tafel scan curves using different scan methods in 450 mL electrochemical cells with CIO 18 working electrode, Pt counter electrode and SCE reference electrode for D. ferrophilus SRB at 3-day incubation (modified from [3]). (Note that the figure is taken from our published journal paper. Do I need to secure copyright permission? Is there a need to include a statement like, (Copyright permitted granted by Elsevier)?.

[0068] FIG. 14 is a graph showing two sets of Tafel curves obtained using two different scan methods in 10 mL test kit containing 7 mL 7-d old D. ferrophilus broth in EASW biotically corroding X65 carbon steel WE with a graphite electrode serving as CE / p-RE, showing severely compressed cathodic curve (i.e., very short vertical voltage range) and much smaller icon-, thus indicating MIC.

[0069] FIG. 15 is a graph showing two sets of Tafel curves obtained using two different scan methods in 10 mL test kit containing 7 mL acetate buffer (pH 3) abiotically corroding X65 carbon steel WE with a graphite electrode serving as CE / p-RE, showing practically the same icoiT values and only small cathodic curve compression, thus indicating abiotic corrosion.

[0070] FIG. 16 is a graph showing two sets of Tafel curves obtained using two different scan methods in 10 mL test kit containing 7 mL deionized water pre-sparged with CO2 (pH 3) abiotically corroding X65 carbon steel WE with a graphite electrode serving as CE / p-RE, showing very close iCOn- values and practically no cathodic curve compression, thus indicating abiotic corrosion.

[0071] FIGS. 17A and 17B are two graphs showing a comparison of (1) a 3-electrode (3E)LPR scan (with SCE RE, X65 WE, graphite CE), in FIG. 17 A, and (2) a 2-electrode (2E)LPR scan (with X65 WE, and graphite CE also serving as pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater), in FIG. 17B.

[0072] FIG. 18 is a graph showing a two-clcctrodc (2E) LPR scan (with X65 WE and 2205 duplex stainless steel alloyed with Cu as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater).

[0073] FIG. 19 is a graph showing a two-electrode (2E) LPR scan (with X65 WE and tantalum as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater).

[0074] FIG. 20 is a graph showing a two-electrode (2E) LPR scan (with X65 WE and 316L as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater).

[0075] FIG. 21 is a graph showing a two-electrode (2E) LPR scan (with X65 WE and Ti as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater), indicating that Ti may not be as good a choice for CE as other materials due to the oscillating curve.

[0076] FIGS. 22A and 22B are two graphs showing Rpvariations of X65 WEs with graphite (a) and Ni (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell A with 250 mL EASW.

[0077] FIGS. 23 A and 23B are two graphs showing Rpvariations of X65 WEs with graphite (a) and Mo (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell B with 250 mL EASW.

[0078] FIGS. 24A and 24B are two graphs showing Rpvariations of X65 WEs with graphite (a) and Cr (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell C with 250 mL EASW.

[0079] FIGS. 25A and 25B are two graphs showing Rpvariations of X65 WEs with graphite (a) and 316L SS (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell D with 250 mL EASW.

[0080] FIGS. 26 A and 26B are two graphs showing Rpvariations of X65 WEs with graphite (a) and CeCh (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell E with 250 mL EASW.

[0081] FIGS. 27A and 27B are schematics illustrating an online biofilm / MIC sensor with two solid-state electrodes and a chamber to allow chemical injections.

[0082] FIG. 28 is a schematic illustrating an online biofilm / MIC sensor with a solid cylinder shaft containing WE and CE / p-RE at its bottom surface.

[0083] FIG. 29 is a graph showing D. ferrophilus in two replicate 10 mL biofilm test kit vials each containing an X65 WE and a graphite CE / p-RE in 5 mL EASW.

[0084] FIG. 30 is a graph showing Rpvariations of X65 WEs in 10 mL, 20 mL, and 50 mL biofilm test kits (with 5 mL, 10 mL, and 25 mL of EASW, respectively) during 7-d incubation with D. ferrophilus .

[0085] FIG. 31 is a graph showing Rpvariations of X65 WEs in 10 mL biofilm test kit vials with different initial inoculum dilution rates during 7-d incubation with D. ferrophilus in 5 mL EASW. (Normal inoculum volume: 100:1 for culture medium to inoculum volume ratio yielding 106cell / mL initial planktonic cell concentration.)

[0086] FIG. 32 is a graph showing Rpvariations of X65 WEs in 10 mL biofilm test kit vials with different initial inoculum dilution rates during 7-d incubation with D. ferrophilus in 5 mL EASW. (Normal inoculum volume: 100:1 for culture medium to inoculum volume ratio yielding 106cell / mL initial planktonic cell concentration.)

[0087] FIG. 33 is a photograph showing an example of a portable potentiostat: A model CS 100 portable potentiostat with a Li-ion rechargeable battery.

[0088] FIGS. 34A and 34B are two photographs showing: (1) In FIG. 34A, a standard 96-well microarray plate, and (2) in FIG. 34B, two wells on a cut-off section of a 24-well plate, each well fitted with a 304 SS WE and a graphite CE / p-RE serving as two biofilm test kit units.

[0089] FIG. 35 is a graph showing Rpcurves of 304 SS WEs from LPR measurements during 7-d incubation of C. difficile with and without vancomycin treatment in two wells on a 24-well plate each containing 1.5 mL BHIS medium at 37°C.

[0090] FIGS. 36A and 36B are two schematics illustrating field deployment of coating sensors: (FIG. 36A) Pre-deployed separate WE attached to the pipeline coating surface and a temporary CE / p-RE placed above WE or near it for electrochemical scans, (FIG. 36B) pipeline metal itself serving as WE with a permanently installed CE / p-RE which can have a protective cover opened only during scans.

[0091] FIG. 37 are photographs showing X65 coupons (1 cm2exposed surface) with different coating conditions used for soil corrosion tests.

[0092] FIG. 38 displays photographs showing vented 450 mL glass bottle containing electrodes before (left) and after (right) being buried in 200 mL sludge.

[0093] FIG. 39 is a photograph showing a large X65 coupon steel plate WE and 316 SS CE / p- RE used in coating disbondment test. (The rectangular box bulge indicates coating disbonded area. The thick vertical line indicates location of an incision line to remove a plastic tape in the “pocket” which allowed sludge and fluid to ingress and come into contact with the bare X65 surface.)

[0094] FIGS. 40A and 40B are graphs showing Rp(FIG. 40A) from LPR measurement, and z'corr (FIG. 40B) from Tafel scans on coated and exposed X65 WEs in 450 electrochemical glass cell containing 200 mL aerobic sludge over 7-d period.

[0095] FIG. 41 is a graph showing Tafel scan curves obtained at 3 d on X65 WE (to simulate external pipe surface) in 450 mL electrochemical glass bottle containing 200 mL sludge.

[0096] FIGS. 42A and 42B are graphs showing variations of Rp(FIG. 42A) from LPR measurement, and zCOrr (FIG. 42B) from Tafel scans in scratching test over 7-d period.

[0097] FIG. 43 is a graph showing LPR vs. time of X65 in coating disbondment test in 450 mL glass bottle containing 200 mL aerobic sludge.

[0098] FIGS. 44A and 44B are graphs showing variations of Rp(FIG. 44A) from LPR measurement, and icon(FIG. 44B) from Tafel scans in scratching test in 450 mL electrochemical glass cell containing X65 WE and 316L SS CE / p-RE buried in 200 mL aerobic sludge over 7-d period.

[0099] FIG. 45 is a photograph showing 10 mL electrochemical glass cells containing 5 mL LB-NO3 medium before incubation with nitrate reducing P. aeruginosa.

[0100] FIGS. 46A and 46B are graphs showing Rp(FIG. 46A) from LPR measurement, and icon (FIG. 46B) from Tafel scans on coated and exposed X65 WEs in 10 mL electrochemical glass cell containing nitrate reducing P. aeruginosa over 7-d period.

[0101] FIGS. 47A and 47B are graphs showing Rpvariations of coated and exposed thin (cold-worked) X65 WEs (FIG. 47A) and exposed normal X65 WE vs. thin (cold-worked) X65 WE (FIG. 47B) in 450 mL electrochemical glass cell containing 200 mL aerobic sludge.

[0102] FIG. 48 is a graph showing Rpcurves of X65 WEs incubated with D. ferrophilus in 10 mL biofilm test kit containing 5 mL EASW with and without THPS biocide treatment.

[0103] FIG. 49 is a graph showing Rpbefore and after tandem injections of MNPs, RF, and biocide THPS at 3 d of incubation with oilfield biofilm Consortium lie

[0027] in 10 mL biofilm test kit containing X65 WE and graphite CE / p-RE in 5 mL EASW at 37°C

[0028] .

[0104] FIG. 50 is a graph showing Rpvariations of 316L Stainless steel (SS) WE in 10 mL biofilm test kit containing 5 mL wet sludge.

[0105] FIG. 51 are photographs showing a miniature electrochemical cell based on a 10 mL serum vial with 2 solid-state electrodes: (a) with a vent (a filter on a needle) for aerobic biofilm growth, and (b) without a vent for anaerobic biofilm growth.

[0106] FIGS. 52A-52D are graphs showing a 10 mL biofilm test kit (304 SS WE and graphite CE / p-RE) used to assess antimicrobial efficacy with 3-day C. difficile incubation in 7 mL BHIS medium before antimicrobial injection: FIG. 52A shows vancomycin injection leading to corrosion rate decrease (Rpincrease) in 3 hours; FIG. 52B shows increased corrosion (decreased Rp) during the 3-day incubation due to biofilm establishment on WE, and corrosion rate decrease (Rpincrease) during the 1 day after injection with FIG. 52A being the hours around 3-day point in FIG. 52B; FIG. 52C shows metformin failure to inhibit pre-established C. difficile biofilm compared to fast biofilm by THPS; and FIG. 52D shows successful metformin prevention of C. difficile biofilm formation.

[0107] FIG. 53 is a graph showing LPR measurement of 304 SS incubated with aerobic P. aeruginosa in 10 mL biofilm test kit containing 5 mL Luria-Bertani (LB) medium at 37°C.

[0108] FIG. 54 is a graph showing Rpcurves from LPR measurements during 7-d incubation of galvanized steel with nitrate reducing P. aeruginosa in 10 mL biofilm test kit containing 5 mL LB-NO3 medium with and without THPS and trehalase for biofilm treatment at 37°C (modified from

[0035] ).

[0109] FIG. 55 is a schematic illustration of biofilm detection on a storage tank using a portable coating (damage) sensor to pinpoint biofilm location.

[0110] DETAILED DESCRIPTION OF THE INVENTION

[0111] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which mayvary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0112] As noted above, there are several drawbacks to current methods and apparatus for detecting biofilms and biocorrosion, for distinguishing different types of corrosion (such as biotic versus abiotic), and for assessing antimicrobial treatment of biofilms and biocorrosion offline and online.

[0113] Currently expensive and large sensors are used. These sensors cannot distinguish biocorrosion type. They cannot distinguish abiotic corrosion from biocorrosion accurately.

[0114] Various aspects of the present invention reduce these drawbacks by providing an apparatus (a test kit or a sensor) with the apparatus being small, low cost, and disposable. It can detect corrosion rate and corrosion type. It can be widely used to provide numerous data on biocorrosion. Currently, microbiology test kits only provide microbe information rather than biocorrosion information. One working principle for the present apparatus (and also online or offline sensors) is based on measuring biofilm corrosivity electrochemically to reflect biofilm growth / health. It can also be used to assess antimicrobial biofilm treatment efficacy.

[0115] Thus, one aspect of the present invention accomplishes this by providing an apparatus including an electrochemical cell including first and second solid-state electrodes. In this embodiment, the first solid-state electrode is a working electrode, and the second solid- state electrode is a counter electrode. In particular, the electrochemical cell of this aspect of the present invention does not include a dedicated and separate third electrode - being a reference electrode - in addition to the first and second solid-state electrodes. Rather, the counter electrode can also function as a pseudo-reference electrode.

[0116] A user can introduce a fluid or liquid suspension of particles for incubation to grow biofilms on the working electrode of the apparatus. Various electrochemical scans maythen be performed to distinguish abiotic corrosion from biocorrosion, and to detect biocorrosion rate.

[0117] In one example of a particular embodiment, the apparatus may be a miniature electrochemical cell. For example, the cell may include as small as a 10 mL (or even a 5 mL) serum vial with two solid-state electrodes and a rubber septum. A 1 cm2(or smaller) metal coupon may be used as working electrode. And a 1 cm2(or smaller) graphite or other conductive material may serve as the counter electrode / pseudo-reference electrode combination. As described above, a user can inject a fluid or liquid suspension of particles for incubation to grow biofilms on the working electrode. Various electrochemical scans may then be performed to distinguish abiotic corrosion from biocorrosion, and to detect biocorrosion rate.

[0118] FIG. 1 shows an example of aparticular embodiment of the inventors’ apparatus (e.g., a disposable biofilm / MIC test kit design) using a standard serum vial (a 10 mL serum vial - i.e., the volume of the interior chamber of the vial, which operates as the housing for the electrochemical cell, is 10 mL) containing two solid-state electrodes (each with working surface area of around 1 cm2, but larger or smaller surface areas may also work if not 10X smaller or larger). In this particular embodiment, one of the solid-state electrodes is a carbon steel working electrode (WE), and the other of the solid-state electrodes is a graphite counter electrode / pseudo-reference electrode (CE / p-RE). A microbial biofilm can attach to the WE surface, but is less likely to attach to the CE / p-RE surface, because the latter is chosen to be a material that is biofouling-resistant. Other materials such as stainless steel (SS) can also be used as WE. There are also other choices for CE / p-RE discussed in greater detail below. The inside of the vial may be sterilized and pre-filled with filter- sterilized nitrogen gas for injection of anaerobic samples. For aerobic samples, a needle fitted with a disposable sterile syringe filter can be used for venting. A 10 mL vial-based test kit is usually injected with 5 mL to 7 mL liquid sample, sometimes with extra nutrients to speed up biofilm growth. For a vial prefilled with nitrogen gas, liquid injection will increase vial pressure. At the end of injection, the needle’s tip should be positioned in the gas headspace to allow excess gas pressure to push the plunger of the syringe upward to release the excess pressure in the vial.

[0119] Although the size of the volume of the interior chamber in the particular embodiment described above (with respect to FIG. 1) is 10 mL, other volumes may be used. Such volumes may range from 1 mL to 200 mL in various embodiments. And particular volumes may include an interior chamber of 5 mL, 7 mL, 15 mL, 20 mL, 30 mL, 50 mL, 100 mL, 125 mL, or 200 mL. FIG. 2 shows two such sizes (20 mL and 50 mL). A smaller vial size is good in minimizing the sample volume and other chemicals used. A bigger vial can be used to allow more microbial cells in the liquid phase which can facilitate biofilm formation on the WE, especially when the liquid is low in nutrients and planktonic microbes.

[0120] As described above, one aspect of the present invention is directed to the use of two solid-state electrodes (one working electrode and one counter electrode) - with the elimination of the need for a separate reference electrode. The classical 3-electrode setup that the present invention avoids is shown in FIG. 3A as including a WE, a CE and a dedicated RE

[0021] , The dedicated RE is needed to obtain accurate OCP. However, a typical RE such as saturated calomel electrode (SCE) and Ag / AgCl (with KC1 electrolyte) electrode requires the use of a liquid electrolyte which makes the device not practical in field applications. They are also too expensive as a disposable test kit.

[0121] For the 2-electrode (2E) apparatus of the present invention for electrochemical testing, the non-solid-state SCE reference electrode is not used, as described above. In certain embodiments, a platinum mesh serves as both counter electrode and pseudo-RE at the same time. FIG. 3B is a photograph of a 450 mL 3E cell after and before SRB incubation. (The large size and a non-solid-state dedicated RE make the 3E device not suitable for field uses.) FIG. 3C is an image of a SCE. FIG. 3D is a schematic of a 2-electrode (2E) electrochemical testing according to aspects of the present invention, where the non-solid-state SCE reference electrode is not used; rather the counter electrode serves as a pseudo-reference electrode (e.g., the platinum mesh serves as both counter electrode and pseudo-RE at the same time).

[0122] Without a dedicated RE, scans performed with a 2-electrode (2E) apparatus (of the present invention) does not provide as reliable OCP readings as 3-electrode (3E) scans. However, OCP is not needed to gauge MIC rates. In fact, a lower OCP theoretically reflects a higher tendency for corrosion (rather than corrosion rate), but it is often misleading in the prediction of MIC outcome due to the complexity of the MIC system [10,22].

[0123] More specifically, As described above, one commonly used electrochemical measurement in corrosion is open circuit potential (OCP), but the inventors discovered that passive measurements such as OCP and microbial fuel cell output measurements for MIC are unreliable and misleading due the complexity of the corrosion system. As noted, OCP is often misleading because a lower OCP theoretically merely means a higher tendency for corrosion rather than corrosion rate. A higher tendency for corrosion can be dampened by slow kinetics. The higher tendency for corrosion may come from a corrosion process that is not responsible for the actual corrosion because it is kinetically too retarded. Instead, a different corrosion process may be responsible, but its OCP may be much higher (i.e., lower tendency for corrosion). Thus, it is not reliable to use OCP to predict corrosion.

[0124] However, the inventors also discovered that non-passive linear polarization resistance (LPR), Tafel (also known as potentiodynamic polarization), electrochemical impedance spectroscopy (EIS), and electrochemical frequency modulation (EFM) are reliable in predicting corrosion trends. Electrochemical scans are performed using a potentiostat (also known as an electrochemical workstation). OCP is a passive measurement method without using an externally imposed electric field. LPR and Tafel scans use an externally imposed DC (direct current) electric field for scanning, while EIS and EFM use AC (alternate current) electric fields. Thus, the inventors have determined that kinetic scans (such as LPR, Tafel, EIS and EFM) should be used instead.

[0125] Thus, there may be various embodiments apparatus including an electrochemical cell including first and second solid-state electrodes, wherein the first solid- state electrode is a working electrode, and the second solid-state electrode is a counter electrode (and functions as a pseudo-reference electrode). In particular, the electrochemical cell may further include a housing and a closure that define an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber, and wherein the closure is adapted to provide access to the interior chamber. An example of a housing and closure is the vial and septum (such as a rubber septum) arrangement described above. In certain embodiments, the interior chamber may include a gas (such as nitrogen gas).

[0126] In certain embodiments, the first solid-state electrode comprises a conductive metal. Such conductive metal may be chosen from iron, carbon steel, stainless steel,galvanized steel, copper, zinc, aluminum, galvanized steel, bronze, brass, titanium, chromium steel, and nickel, and various other metals and alloys when their corrosion needs to be investigated (for example, if a pipeline is being tested, and is made of a certain material, the first solid-state electrode may include that material - or be coated with that material - such that the working surface of the electrode is of the same material as the pipeline, or other component being tested). As has been described herein, the apparatus may be used to detect and / or assess biofilms / biocorrosion that is occurring in or on a separate component (for example, such component may be a conduit, such as a pipeline). That separate conduit will be made of a particular material or materials - and so, in certain embodiments, the material used for the first solid-state electrode (the working electrode) can be selected to match the material of the separate component (e.g., pipeline) that is being tested.

[0127] The second solid-state electrode, in various embodiments, comprises a material chosen from graphite, a conductive metal, a conductive polymer, a conductive organic material, a conductive inorganic material, a coated metal, and a coated polymer. In certain embodiments, where the material is a conductive metal, the conductive metal may be chosen from copper, platinum, tantalum, nickel, molybdenum, chromium, cerium (or cerium oxide), steel, stainless steel, and brass.

[0128] As noted above, an apparatus (such as the apparatus of the various embodiments described above) can be used to detect biocorrosion in the area of pipelines (although the use of the apparatus would not be limited to pipelines, such as oil or gas pipelines) - but could be applicable to any conduit or storage vessel, or indeed to any component that houses or transports a fluid - or is simply subject to corrosion.

[0129] The material of the second solid-state electrode may be a material such as plastic or glass. In such embodiments, the material may then be coated, with a conductive or semi-conductive material (such as cerium oxide or platinum).

[0130] Thus, another aspect of the present invention is directed to a system for the detection of biofilms and biocorrosion, for the assessment of biofilms and biocorrosion, and for the assessment of treatment of same, the system comprising a component that is potentiallysubject to biocorrosion or to biofilms associating therewith, and an apparatus (including electrochemical cell) as described above.

[0131] The ’’component,” then, may be a structure that includes an interior space (such interior space capable of containing a material - such as a fluid). And the apparatus is one that includes an electrochemical cell including first and second solid-state electrodes, wherein the first solid-state electrode is a working electrode, and the second solid-state electrode is a counter electrode. The apparatus, then, may not include a dedicated and separate third electrode - being a reference electrode - in addition to the first and second solid-state electrodes. Rather, the counter electrode can also function as a pseudo -reference electrode.

[0132] In one aspect, the apparatus may be a stand-alone apparatus (i.e., separate from the component, e.g., pipeline or other conduit). The material of the working electrode of the apparatus may be of the same material that forms the component (e.g., pipeline or other conduit). A sample of fluid from the pipeline can then be introduced into the electrochemical cell (such as via injection through a septum that closes the housing of the electrochemical cell. Various electrochemical scans may then be performed to distinguish abiotic corrosion from biocorrosion, biocorrosion types, and to detect biocorrosion rate.

[0133] In another aspect, the apparatus may be associated with the component (e.g., pipeline or other conduit), such as by being affixed thereto and allowing for direct introduction of electrodes to fluid in the component, or introduction of fluid into electrochemical cell. For example, part of the housing of the electrochemical cell may be contiguous with a wall of the pipeline. There may be a valve between the pipeline and cell. And the valve may allow for the first and second electrodes of the electrochemical cell to pass through the valve into the interior space of the pipeline to contact fluid therein. The electrodes may then be retracted through the valve and back into the electrochemical cell. Additionally, or alternatively, the valve may allow for fluid from the pipeline to pass into the cell when the valve is opened.

[0134] In addition to detecting, assessing, and assessing treatment of biofilms and corrosion (such as biocorrosion) interior to a component (such as a pipeline), another aspect of the present invention is directed to detecting, assessing, and assessing treatment of biofilmsand corrosion (such as biocorrosion) on the external surface of a component (such as a pipeline), or to a coating on such external surface.

[0135] Thus, in certain embodiments, the electrochemical cell of the apparatus may include a housing that defines an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber when in a first position, and the first and second solid-state electrodes being moveable to a second position positioned outside of the interior chamber. At least one valve may be associated with the housing, wherein the first and second solid-state electrodes are positioned on one side of the valve when in the first position, and wherein the first and second solid-state electrodes are positioned on an opposite side of the valve when in the second position.

[0136] In another embodiment of the system, there may be a second valve associated with the housing of the apparatus. This second valve is adapted to allow for the introduction of materials into the interior chamber of the electrochemical cell of the apparatus.

[0137] Apart from the apparatus and system including apparatus discussed above, as noted previously, coatings are used to protect various metal surfaces against corrosion / biocorrosion. They can be damaged by scratches and disbondment. Corrosive chemicals and microbes can access the metal and cause corrosion / biocorrosion. Thus, as noted above, there is a need to monitor damages to coatings on various assets such as industrial, marine, and military assets.

[0138] In this aspect of the present invention, an apparatus may be used similar to that described above with respect to testing the interior of various components. In that regard, then, this aspect of the present invention may include providing an apparatus including an electrochemical cell including first and second solid-state electrodes. In this embodiment, the first solid-state electrode is a working electrode, and the second solid-state electrode is a counter electrode. In particular, the electrochemical cell of this aspect of the present invention does not include a third electrode - being a reference electrode - in addition to the first and second solid-state electrodes. Rather, the counter electrode can also function as a pseudoreference electrode.

[0139] In this aspect, the present inventors adopt solid-state electrodes (as disclosed above) to monitor corrosion resistance and corrosion rate using electrochemical methods including linear polarization, potentiodynamic polarization and electrochemical impedance spectroscopy scans. The working electrode (WE) can be the base metal under the coatings or a more easily corroded metal covered with (to be damaged) coatings. The counter electrode / pseudo-reference electrode (CE / p-RE) (combination electrode) can be graphite, a conductive polymer stainless steel and other metals (e.g., metal containing platinum, cerium, chromium, nickel). The electrodes have a small working surface (as small as 1 cm2 or less). The WE and CE / p-RE are solid-state materials. The two electrodes are connected to a potentiostat to perform the electrochemical scans periodically. If corrosion rate becomes significant (or corrosion resistance drops significantly), it means the coatings are damaged.

[0140] In certain embodiments of the apparatus that are used both internally and externally to a component (such as a pipeline) only solid-state electrodes are used. Because of the use of solid-state electrodes, no electrolyte-filled reference electrode is needed. Additionally, the electrodes that are used can be quite inexpensive (for example, even Pt coated electrodes are fairly inexpensive). These inexpensive solid-state electrodes can be used in numerous places on an asset such as a pipeline's external surface which is prone to soil or sludge or polluted water corrosion (with and without microbes to contribute to the corrosion) once coatings are damaged.

[0141] Due to its small size, the sensor can be portable / mobile to detect damages in coatings in different places, or an online sensor can be deployed at a fixed location. Additionally, due to the determination by the inventors that even inexpensive electrodes can work effectively (and in such a small apparatus), the apparatus is not only portable, but its solid-state electrodes can also be disposable (though they can also be reused if cleaned and sanitized).

[0142] Due to the nature, materials, size, etc. of the apparatus of the present invention, the apparatus provides fast scans (e.g., as fast as 2 minutes) and are able to provide transient corrosion data to indicate damages in coatings that lead to corrosion. Further, the apparatus is capable of identifying disbondment (indicated by a large corrosion rate increase detected by our sensor) in addition to scratches. The apparatus can be easily embedded on buried pipelines.It can be used when submerged in field liquid such as agricultural runoff, rain water or sewer water. And it can be used to distinguish abiotic corrosion from biocorrosion and biocorrosion type after coatings are damaged (as will be described in greater detail below).

[0143] Thus, as described above, the present inventors have determined that kinetic scans (LPR, Tafel, EIS and EFM) should be used instead of passive OCP. Among these electrochemical methods, LPR is the fastest. Each scan normally takes 2 min to complete, and it can be reduced to 1 min if needed. Tafel, EIS and EFM scans take about 10 times longer (but are still better and more reliable than the use of OCP, as described above). Among these methods, EIS is particularly useful in testing integrity of coatings

[0020] .

[0144] Another aspect of the present invention is directed to the use of the apparatus described herein to test the antimicrobial efficacy of a medical biofilm. It can be used in assessing the growth of environmental biofilms that cause biofouling and biocorrosion, as well as assessing the efficacy of various treatments against these biofilms.

[0145] As noted above, current methods and apparatus for assessing antibiotics efficacy against medical biofilms that cause diseases are tedious and the efficacy data generated tend to have large error bars. The use of the apparatus of the present invention is relatively simple, and can provide transient information on efficacy that is difficult to obtain using existing methods. Further, the data generated in the apparatus, systems, and methods of the present invention, result in less uncertainties in judging efficacy because the same biofilm can be continuously monitored before and after biocide treatment without using two separate biofilms (one as untreated control, and the other treated).

[0146] As discussed above in the Background section, antimicrobial efficacy testing usually is done by enumerating sessile cells in biofilms with and without antimicrobial treatment. To enumerate sessile cells in biofilms, sessile cells are scraped off a solid surface and re-suspended in water or a buffer. Motile bacterial cells can be counted rather accurately using a hemocytometer at 400X magnification because the very small cell dots can be differentiated from artifacts. In antimicrobial treatment studies, a cell suspension’s cell count may be well below the 50,000 cells / mL threshold for accurate hemocytometer counting (of motile bacterial cells) or other imaging methods with stains such as CLSM (confocal laserscanning microscopy) under 400X microscope, because cells are rarely seen in the view field (i.e., there are not enough of them to be seen). Using 40X magnification can cover a 100X larger view area than 400X magnification, but the bacterial cells are too small to be identifiable even for motile cells or stained cells. The Most Probable Number (MPN) serial dilution method can be used to grow cells for counting. The Colony Forming Unit (CFU) plating method can also be used to grow colonies for counting. However, as is well known by those of ordinary skill in the art, these methods are time-consuming and not accurate. Cell counts in medical biofilm studies often carry wide error bars, making statistical significance hard to achieve in many cases.

[0147] The apparatus in various aspects of the present invention, however, makes testing easy. A user only needs to introduce sample into the apparatus (e.g., a 10 mL serum vial with two solid-state electrodes) of the present invention (e.g., by making injections into the electrochemical cell described herein), and then perform electrochemical scans. The methods described herein provide more data [such as minimum dosage, time to kill, kill persistence (time for biofilm to recover), prevention persistence, and efficacy level (reflected by corrosion rate reduction)]. Additionally, multiple apparatus can be run at the same time to assess multiple samples.

[0148] Another aspect of the present invention includes a system for the detection of biofilms and biocorrosion, for the assessment of biofilms, biocorrosion and biofouling, and for the assessment of treatment of same, wherein a surface of the component to be tested is used as the working electrode itself. In an embodiment of such a system, the system includes (1) a conductive component that is potentially subject to biocorrosion or to biofilms associating therewith wherein the component comprises a metal, and (2) a solid-state electrode. A surface of the component is used as a working electrode, and the solid-state electrode is used as a counter electrode, such that the surface of the component and the solid-state electrode comprise an electrochemical cell.

[0149] In various embodiments of this system, the electrochemical cell may further comprise a liquid in contact with the working electrode and the solid-state electrode.

[0150] As noted above, a surface of the component itself may be used as the working electrode. The component itself may be as described above. For example, the component may include an interior space adapted to contain at least one fluid therein. Thus, the component may be a conduit for transport of the at least one liquid. In various examples, the conduit may be a pipeline or a storage tank. In other embodiments, the component may be a metal pillar exposed to a liquid including water or a liquid suspension of particles including water.

[0151] The solid-state electrode in this aspect of the invention may include a material chosen from graphite, a conductive metal, a conductive polymer, a conductive organic material, a conductive inorganic material, a coated metal, and a coated polymer. In certain embodiments where the material is a conductive metal, the conductive metal is chosen from stainless steel, copper, platinum, tantalum, nickel, molybdenum, chromium, steel, and brass.

[0152] Alternatively, the material material a plastic, ceramic or glass - and may be coated with a conductive or semi-conductive material. For example, the material may be a coated metal, ceramic, or coated polymer, (or other material) wherein the metal, ceramic, or polymer (or other material) is coated with cerium oxide or platinum.

[0153] Another aspect of the invention may include a method for detecting biofilms and biocorrosion, for distinguishing types of corrosion, or for assessing antimicrobial treatment of biofilms and biocorrosion. The method may include the steps of (1) obtaining a sample from a component (such as those described above) having at least one surface subject to corrosion; (2) introducing the sample to an apparatus including an electrochemical cell comprising first and second solid-state electrodes, wherein the first solid-state electrode is a working electrode and the second solid-state electrode is a counter electrode (wherein the electrochemical cell does not include a dedicated reference electrode in addition to the first and second solid-state electrodes); and (3) testing the working electrode to determine if a biofilm has grown on the working electrode. At least the working electrode may be incubated for a period prior to testing. Such an incubation period may be for a day or days.

[0154] As with other aspects of the invention (above), the sample may be a liquid or a liquid suspension of particles.

[0155] Further, in embodiments of this aspect, the component may have a defined interior space, and the sample is obtained from the interior space of the component. The at least one surface of the component may be a surface that faces the interior space of the component, such that any sample in the interior space is capable of contacting the at least one surface. As above, the component may a conduit for transport of the at least one fluid. The component may be a pipeline, a storage tank, a metal wall or a metal pillar exposed to a fluid containing water.

[0156] The apparatus used in embodiments of this method may further comprise a housing and a closure that define an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber, and wherein the closure is adapted to provide access to the interior chamber. The apparatus used may be that described above. Thus, the closure may be a septum, and introducing the sample to the apparatus may comprise injecting the sample through the septum of the apparatus.

[0157] Alternatively, in another embodiment, introducing the sample may comprise moving at least the two solid-state electrodes from the interior chamber of the apparatus into the interior space of the component. And the method may then further comprise subsequently moving the solid-state electrodes from the interior space of the component back into the interior chamber of the apparatus.

[0158] In certain embodiments, the apparatus and component are adjacent one another, and further comprise a valve that allows for communication between the interior chamber of the apparatus and the interior space of the component, wherein moving the solid-state electrodes comprises opening the valve to allow the solid-state electrodes to pass from the interior chamber to the interior space, or to retract from the interior space to the interior chamber.

[0159] In other embodiments, introducing the sample comprises causing at least a portion of the sample to move from the interior space of the component to the interior chamber of the apparatus. In a particular embodiment, the apparatus and component are adjacent one another, and further comprising a valve that allows for communication between the interior chamber of the apparatus and the interior space of the component, and wherein opening thevalve allows bulk fluid and the solid-state electrodes to move from the interior space to the interior chamber.

[0160] In certain embodiments, the at least one surface is an external surface of the component, and at least a portion of the external surface is used as the working electrode.

[0161] In embodiments of the method, testing the working electrode further comprises performing an electrochemical scan chosen from open circuit potential, linear polarization resistance, potentiodynamic polarization, electrochemical impedance spectroscopy, and electrochemical frequency modulation. Further, a portable or bench-top potentiostat may be operatively connected to the apparatus, and the potentiostat is used in the electrochemical scan. Results of any scan may include data for, or related to, corrosion rate, inverse of corrosion resistance, or corrosion rate trend.

[0162] In the method, one may then correlate biofilm growth or efficacy of a biocide from the corrosion rate, from the inverse of corrosion resistance, or from the corrosion rate trend.

[0163] In another embodiment, the method may further comprise introducing a biocide and an electron mediator into the interior chamber of the apparatus. In certain embodiments, the electron mediator is introduced before introduction of the biocide. The biocide may include, but is not limited to, tetrakis-hydroxymethyl phosphonium sulfate (THPS), glutaraldehyde, alkyldimethylbenzylammonium chloride (ADBAC), 2,2-dibromo-3- nitrilopropionamide (DBNPA), or anthraquinone.

[0164] The biocide may be an antibiotic, and the antibiotic may chosen from, but is not limited to, vancomycin, amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, and levofloxacin.

[0165] The electron mediator may be riboflavin (or other flavins), and / or magnetite nanoparticles. Testing the working electrode may then further comprise performing at least one potentiodynamic polarization scan. For example, one may determine whether anycorrosion is biotic corrosion or abiotic corrosion, or determine type of microbiologically induced corrosion, based on the results of at least one potentiodynamic polarization scan.

[0166] Another aspect of the present invention is directed to a method for detecting biofilms or assessing antimicrobial treatment of biofilms, in vivo. The method may include: (1) inserting first and second solid-state electrodes into the intestines or respiratory tract of a mammal, wherein the first solid-state electrode is a working electrode and the second solid- state electrode is a counter electrode; and (2) testing the working electrode to determine if a biofilm has grown on the working electrode, or to assess treatment of a biofilm.

[0167] The above disclosed aspects and principles of the present invention (related to the apparatus, system, and use) will be further described with respect to the Examples below.

[0168] EXAMPLES

[0169] EXAMPLE 1

[0170] This example is directed to demonstrating principles of the present invention as they relate to the use of the apparatus, systems, and methods described herein with respect to use of an apparatus including first and second solid-state electrodes (working electrode and counter electrode / pseudo-reference electrode).

[0171] 1. A dedicated reference electrode is not necessary in providing corrosion trends

[0172] FIG. 4 shows that the general trend of polarization resistance (Rp) from LPR for 2E is very similar to that from 3E in nitrate-reducing Pseudomonas aeruginosa MIC of C1018 carbon steel which is accelerated by electron mediator riboflavin

[0023] , Note that the inverse of Rpreflects corrosion rate. In summary, then, FIG. 4 shows LPR scans with and without RE (3- electrode system vs. 2-electrode system) both showing increased MIC of C1018 carbon steel after electron mediator (riboflavin) injection into 3-d nitrate -reducing P. aeruginosa broth in a 450 mL glass cell with 250 mL Luria-Bertani medium with KNO3 (LB-NO3) culture mediumat 37°C, proving that a dedicated RE (third electrode) is not essential in providing corrosion trends.

[0173] 2. LPR, Tafel, EIS, and EFM scans using the apparatus, systems, and methods of the present invention

[0174] FIG. 5 shows LPR monitoring of SRB MIC of carbon steel during 7-d incubation. In particular, FIG. 5 is a graph showing variations of X65 WE Rpduring 7-d incubation of D. ferrophilus in 5 mL EASW in 10 mL biofilm test kit at 28°C. (Range bar obtained from two replicate 10 mL electrochemical glass cells.) At 0 d right after injecting 0.5 mL SRB inoculum into 5 mL enriched artificial seawater (EASW) culture medium [9], RPwas high (corrosion rate low). However, once biofilm started to grow, Rpstarted to decrease (corrosivity increased). At 3 d, Rpleveled off, which indicates biofilm maturity at 3 d. After that, the biofilm’s corrosivity did not change much. In summary, the data shown in FIG. 5, then, demonstrates that electrochemically measured corrosivity can be used to monitor biofilm growth via corrosivity change in a 10 mL electrochemical glass cell (biofilm test kit). The decrease in corrosion resistance Rpindicated increased corrosion rate in the first 3 d. Rpleveled off after 3 d, indicating biofilm maturity without further corrosivity increase.

[0175] FIG. 6, FIG. 7, and FIG. 8 show some examples of different electrochemical scans. In particular, FIG. 6 is a graph showing corrosion current density (Lorr) values fitted from Tafel scans during 7-d incubation of D . ferrophilus in 5 mL EASW in 10 mL biofilm test kit. FIG. 7 is a graph showing corrosion rate trend based on EFM results during 7-d incubation of D . ferrophilus in 5 mL EASW in 10 mL biofilm test kit. And FIG. 8 is a graph showing variations of (Rf + Rct) (corrosion resistance equivalent to Rpin LPR) of X65 WE fitted from EIS data during 7-d incubation of D. ferrophilus in 5 mL EASW in 10 mL biofilm test kit. In EIS, Rf and Rct denote the resistance of the combined biofilm and corrosion product film, and charge transfer resistance. They are calculated from modeling EIS spectra using an equivalent circuit model

[0010] . The Rf + Rct from EIS is often used to compare with Rpfrom LPR. In summary, FIG. 6, FIG. 7, and FIG. 8 proved that corrosion current density from Tafel scans, corrosion rate from EFM scans, and corrosion resistance from EIS are equally capable of indicating biofilm growth and predicting biofilm maturity time as LPR. However, these scans tend to take 10X longer time than LPR scans as discussed above.

[0176] FIG. 9 shows that after a D. ferrophilus biofilm reached maturity at 3 d, tandem injections were made to identify MIC type and biocide efficacy. FIG. 9 is a graph of an LPR scan showing increased corrosion after electron mediator (riboflavin) injection (into 3-d old broth), and decreased corrosion after THPS biocide injection for SRB MIC of X65 pipeline steel by D. ferrophilus (a very corrosive SRB strain) in a 10 mL biofilm test kit containing 7 mL EASW (enriched artificial seawater) at 28°C. More specifically, in the figure, 0 min corresponds to 3 d incubation time point. FIG. 9 shows that 10 min after the injection of riboflavin electron mediator, the SRB corrosion rate increased considerably. This suggests that SRB MIC of carbon steel belongs to EET-MIC because riboflavin accelerated EET

[0024] . The subsequent tetrakis-hydroxymethyl phosphonium sulfate (THPS) biocide injection caused a large decrease in corrosion rate, which means that THPS mitigated the biofilm. The Rpincrease of 29% can be used as an indicator for THPS biofilm treatment efficacy based on corrosion inhibition efficiency. In summary, FIG. 9 shows that the 10 mL electrochemical cell meaningfully described the SRB EET-MIC acceleration by riboflavin electron mediator, and corrosion rate decrease after injection of THPS (tetrakis hydroxymethyl phosphonium sulfate) biocide as expected. Thus, the injection of an electron mediator into the biofilm test kit can be used to distinguish EET-MIC from other types of MIC and abiotic corrosion. Furthermore, the injection of a biocide can be used to assess biocide efficacy.

[0177] The inventors recently demonstrated that repeated Tafel scans are permitted if the scans are initiated from OCP (open circuit potential) to - 200 mV (vs. OCP), and from OCP to +200 mV (vs. OCP) separately to obtain cathodic and anodic curves separately, rather than the harsher scan method from -200 mV (vs. OCP) to+200 mV (vs. OCP) upward continuously, which allows no time for the biofilm to adapt to an external voltage

[0040] , FIG. 10 shows a corrosion current density (jeon- or icon- in this document) time profile from Tafel scans on several days for MIC of X65 pipeline steel by D. ferrophilus SRB in a 10 mL miniature 2E electrochemical cell containing 7 mL enriched artificial seawater. FIG. 10 has the same experimental condition as FIG. 6, except the injection volume difference (7 mL in FIG. 10 vs. 5 mL in FIG. 6). The D. ferrophilus SRB biofilm usually takes 3 or 4 days to reach maturity. FIG. 10 shows that its jCOrr peaked at day 4. Biofilm aggressiveness may be correlated using jeorr data directly in MIC modeling. In summary, Fig. 10 once again (like FIG. 6) showscorrosion current density from Tafel scans can be used to judge biofilm growth and biofilm mature.

[0178] Abiotic corrosion is not caused by a biofilm. Thus, it cannot be accelerated by injection of an electron mediator such as riboflavin, or decreased by biocide injection, in contrast to how biotic corrosion by a biofilm behaves as shown in FIG. 9. FIG. 11 shows the LPR scan profile of 10 mL test kit containing 7 mL 5% (w / w) NaCl solution pre-sparged with CO2 (pH 4) abiotically corroding X65 carbon steel WE with a graphite electrode serving as both CE and pseudo-RE. The Rpprofile shows injection disturbance (an uptick right after injection. However, riboflavin (RF) electron mediator injection did not increase the CO2 corrosion (i.e., did not decrease Rp). Furthermore, THPS biocide injection did not decrease CO2 corrosion. FIG. 12 is a repeat run of FIG. 11. Both FIG. 11 and FIG. 12 show injection disturbance, but not increased corrosion for riboflavin (RF) electron mediator injection or decreased corrosion after THPS biocide injection, thus supporting abiotic corrosion. In summary, FIG. 11 and FIG. 12 demonstrate that electron mediator and biocide injections can also be used to distinguish abiotic corrosion from EET-MIC because abiotic corrosion does not respond to the injections with corrosion rate changes. Note that riboflavin can accelerate EET-MIC, but not M-MIC such as biogenic sulfide corrosion of copper

[0041] .

[0179]

[0180] Thus, biocide injection can be used to distinguish abiotic corrosion from MIC. It is also desirable to distinguish abiotic corrosion from MIC using electrochemical scans without injecting a biocide. The inventors discovered that in Tafel scans of MIC systems, the continuous upward scan from lowest voltage to highest voltage (usually from -200 mV vs. OCP to +200 mV vs. OCP) will show Tafel skews, when compared with the more accurate and gentle dual-half scans from OCP to -200 mV vs. OCP for the cathodic curve, and from OCP to +200 mV vs. OCP for the anodic curve as shown in FIG. 13 from

[0025] . The harsh continuous upward scan tends to compress the cathodic curve (in terms of shrinking voltage range) while elongating the anodic curve (FIG. 14). More importantly, it yields a much lower (e.g., 50% lower) iCOrr compared to the dual-half scan. This is attributed to the fact that continuous upward scan applies the most negative voltage without allowing any time for the biofilm to adapt to the externally imposed voltage. In dual-half scans, both cathodic and anodic curves start at OCP (at which there is no external voltage), and then departs to reach -200 mVvs. OCP (for cathodic curve), or to +200 mV vs. OCP (for anodic curve). This means the biofilm on the WE has time to adapt. Abiotic corrosion has no biofilm on the WE. Thus, Tafel skews are quite minimal. FIG. 15 and FIG. 16 show that in abiotic acetic acid corrosion and CO2 corrosion in the absence of oxygen, no obvious Tafel skews arc exhibited. Thus, Tafel skews can be used to distinguish MIC from abiotic corrosion.

[0181] FIG. 13 shows Tafel scan curves using different scan methods in 450 mL electrochemical cells with C1018 working electrode, Pt counter electrode and SCE reference electrode for D. ferrophilus SRB at 3-day incubation

[0040] . It shows that the harsh continuous upward scan distorted the Tafel curves because it started with the most negative voltage leaving no time for the biofilm to adapt to the external voltage. Its distorted corrosion current density jeon was found -50% smaller than the value from the gentle cathodic and anodic scans both starting from OCP (i.e., no external voltage position)

[0040] . The gentle scan method is called dual half-scans method. This Tafel scan abnormality is largely absent from abiotic Tafel scans that do not involve living biofilms as shown in FIG. 15.

[0182] FIG. 14 is a graph showing two sets of Tafel curves obtained using two different scan methods in 10 mL test kit containing 7 mL 7-d old D. ferrophilus broth in EASW biotically corroding X65 carbon steel WE with a graphite electrode serving as CE / p- RE, showing severely compressed cathodic curve (i.e., very short cathodic vertical voltage range) and much smaller iCOrr. In summary, FIG. 14 confirms that biotic corrosion by a biofilm suffers very large distortion (severe compression of the cathodic curve) and much smaller corrosion current density jCOrr in continuous upward Tafel scans.

[0183] FIG. 15 shows two sets of Tafel scan curves using two different scan methods in a 10 mL test kit containing 7 mL acetate buffer (pH 3) corroding X65 carbon steel WE with a graphite electrode serving as both CE and pseudo-RE. Although there is small compression (i.e., shorter voltage range) of the continuous upward scan’s cathodic curve (lower half curve), the jCOir values for both dual half-scans (gentle scan method) and continuous upward scans (harsh scan method) are practically the same. Abiotic CO2 corrosion Tafel scans in FIG. 16 indicate very close jeon values for both scan methods and practically no cathodic curve compression. The abiotic corrosion observations are very different from the very large skews seen in FIG. 13 and often 50% lower jCon- for continuousupward scans in biotic systems because biofilms are susceptible to the harsh continuous upward scan method that starts with the most negative voltage instead of zero voltage (vs. OCP) in dual half-scans

[0040] ,

[0184] Similarly, FIG. 16 is a graph showing two sets of Tafel curves obtained using two different scan methods in 10 mL test kit containing 7 mL deionized water pre-sparged with CO2 (pH 3) abiotically corroding X65 carbon steel WE with a graphite electrode serving as CE / p-RE, showing very close icon- values and practically no cathodic curve compression.

[0185] In summary, the data in FIG. 14, FIG. 15 and FIG. 16 combined together prove that Tafel scan skews and much lower corrosion current density jcorr can be used to distinguish MIC from abiotic corrosion because abiotic corrosion does not incur Tafel scan skews and much lower jCOrr when continuous upward scan is used instead of dual half-scans.

[0186] 3. Other choices for use as the solid-state CE / pseudo-RE

[0187] As described above, graphite is one option of conductive material that can be used for the second solid-state electrode (i.e., the counter electrode / pseudo-reference electrode). And, graphite has succeeded in the inventors’ numerous tests as a solid-state CE / pseudo-RE combo for apparatus (biofilm / MIC sensor) in accordance with principles of the present invention. FIGS. 17A and 17B show LPR scans for D. ferrophilus MIC against X65 carbon steel in a 450 mL electrochemical cell.

[0188] FIGS. 17A and 17B are two graphs showing a comparison of (1) a 3- electrode (3E) LPR scan (with SCE RE, X65 carbon steel WE, graphite CE), in FIG. 17A, and (2) a 2-electrode (2E) LPR scan (with X65 carbon steel WE, and graphite CE also serving as pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater), in FIG. 17B. Without SCE as a dedicated RE, the Rpprofile (as seen in the Rpreading in FIG. 17B) is slightly higher, but it predicted biofilm maturity at 2 d well with a similar Rpcurve shape, because in both FIG, 17A and FIG. 17B, the corrosion resistance Rpprofiles decreased and started to level off at 2 d, indicating biofilm maturity at 2 d with no further corrosivity increase.

[0189] Thus, the inventors found that graphite works well as CE / p-RE just like expensive Pt sheets or meshes using in route lab electrochemical tests to study MIC. However, the inventors also desire to have additional and inexpensive choices of solid-state electrodes for CE / p-RE. They may perform equally well or even better in some conditions against biofouling which would impedes an CE / p-RE electrode’s performance. Another consideration is to have more choices when the inventors’ solid-state electrodes are printed as thin films [29,30] using a printer such as a 3D printer.

[0190] A good solid-state electrode choice for an apparatus as described herein should be inexpensive, stable, and resistant to corrosion and biofouling. And so, several metals were tested by the inventors to determine suitability for use in apparatus of the present invention. FIG. 18 is a graph showing two-electrode (2E) LPR scan (with X65 WE and 2205 duplex stainless steel alloyed with Cu as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater). This figure shows that 2205-Cu duplex stainless steel (3% Cu by mass)

[0042] worked fine in predicting biofilm maturity at 3 d. This special stainless steel contains biocidal Cu element against microbes such as Pseudomonas aeruginosa

[0042] , but Cu is effective against SRB because biogenic sulfide can remove biocidal Cu ions extremely well

[0041] . Thus, Cu addition to the stainless steel will not add extra biocidal benefit apart from the biocidal elements such as nickel and chromium already in a common stainless steel like 304 stainless steel.

[0191] FIG. 19 is a graph showing two-electrode (2E) LPR scan (with X65 WE and tantalum as CE / p-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater). Tantalum is highly corrosion resistant. FIG. 19 shows that with it serving as CE / p-RE electrode, the Rpcurve shape is correct. However, it indicates biofilm maturity at 4 d rather than 2 d or 3 d.

[0192] FIG. 20 is a graph showing two-electrode (2E) LPR scan (with X65 WE and 316L as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater). FIG. 20 shows that 316L stainless steel worked reasonably well in predicting the biofilm maturity time at 3 d. However, the Rpcurve is not as stable as that for graphite (FIG. 17).

[0193] FIG. 21 is a graph showing two-electrode (2E) LPR scan (with X65 WE and Ti as CE / pseudo-RE) in 450 mL glass cell (with 250 mL D. ferrophilus culture in enriched artificial seawater). Titanium is very corrosion resistant metal. However, SRB are known to grow a robust biofilm on it

[0043] , because Ti is not biocidal. FIG. 21 indicates that its Rpcurve is not meaningful for MIC trends. Thus, Ti may not be as good a choice for CE / po-RE as other materials tested.

[0194] FIGS. 22A-B, FIGS. 23A-B, FIGS. 24A-B, and FIGS. 26A-B show that Ni, Mo, Cr, 316L SS, CcCL are possible choices for CE / p-RE. And FIGS. 25A-B show that 316L SS is a possible choice for CE / p-RE reiterating that demonstrated by FIG. 20 (above). Other corrosion-resistant metals and conductive polymers can also be considered. They should be biofouling resistant and corrosion-resistant to minimize interference with the electrochemical scans. These materials involve heavy metals that inhibit microbes to certain degrees.

[0195] More specifically, FIGS. 22A-B are graphs showing Rpvariations of X65 WEs with graphite (a) and Ni (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell A with 250 mL EASW. FIGS. 23A-B are graphs showing Rpvariations of X65 WEs with graphite (a) and Mo (b) as CEs / p-REs during 7 -d incubation with D. ferrophilus in 450 mL electrochemical glass cell B with 250 mL EASW. FIGS. 24A-B are graphs showing Rpvariations of X65 WEs with graphite (a) and Cr (b) as CEs / p-REs during 7-d incubation with D. ferrophilus in 450 mL electrochemical glass cell C with 250 mL EASW. FIGS. 25A-B are graphs showing Rpvariations of X65 WEs with graphite (a) and 316L SS (b) as CEs / p-REs during 7-d incubation with D . ferrophilus in 450 mL electrochemical glass cell D with 250 mL EASW. And FIGS. 26A-B are graphs showing Rpvariations of X65 WEs with graphite (a) and CeCh (b) as CEs / p-REs during 7-d incubation with D . ferrophilus in 450 mL electrochemical glass cell E with 250 mL EASW. Cerium oxide is known to be biocidal and is particularly suitable against SRB [7], Thus, cerium oxide coated metals may also be useful. Some other metals such as brass, and conductive polymers can also be used as CE / p-RE.

[0196] EXAMPLE 2

[0197] This example describes various ways that the apparatus and system of the present invention described herein may be used.

[0198] 1. Use of the apparatus including electrochemical cell to get field data and how to use the field data for modeling

[0199] An apparatus (according to the principles of the present invention) will be prefabricated with a WE using the same steel as the pipeline steel, and a solid-state CE / p-RE (e.g., graphite electrode) inserted into a standard 10 mL serum vial. This is labeled as a disposable biofilm test kit. The apparatus is pre-sterilized and filled with N2. About 7 mL of field water or a pig-trap sludge suspension will be injected into each apparatus using a disposable syringe before incubation. To speed up microbial growth, nutrients may be injected into the apparatus to simulate worst-case scenario. Various electrochemical scans such as LPR, EIS, potentiodynamic polarization (i.e., Tafel) scans will be performed by hooking up the two wires on the 10 mL vial (FIG. 1) with a standard potentiostat equipped with corrosion analysis software. The electrochemical cells after injection can be either mailed to a service provider (such as Ohio University MIC research group), or scanned by the end user (such scans may also be tutored by a service provider remotely via the internet). The various scans can provide information to discern MIC rates, MIC types, and biocide treatment efficacy as explained below:

[0200] The electrochemically measured corrosion rate information such as jCorr from Tafel scans can be correlated with weight loss, and used for calibration of biofilm aggressiveness for use by MICORP (MIC prediction software created by Ohio University’s MIC group). This means the use of the test kit and MICORP modeling can form an integrated approach for MIC monitoring and model and prediction.

[0201] Biocide injection response can be used to distinguish abiotic corrosion from MIC because abiotic corrosion (e.g., acetic acid corrosion and CO2 corrosion) does not show a large Tafel curve skew and a large corrosion current density reduction. Tafel scans can also be used as discussed above (FIG. 14, FIG. 15 and FIG. 16). To distinguish EET-MIC (e.g., SRB MIC of carbon steel) from abiotic corrosion, an electron mediator (e.g., riboflavin) can be injected. It will accelerate EET-MIC (FIG. 9), but not abiotic corrosion (FIG. 12).

[0202] EET-MIC and M-MIC can be distinguished by injecting an electron mediator such as riboflavin. EET-MIC will be accelerated while M-MIC will not

[0040] . Furthermore, M- MIC such as APB MIC typically produces extra amount of headspace Fh, which can be detected from the electrochemical cells using a handheld Eh sensor or gas chromatography. Typically, the headspace EE sample needs to be diluted using air in an anaerobic bottle because the Eh sensor is very sensitive (0 to 999 ppm)

[0041] , EET-MIC, such as carbon steel MIC caused by D. vulgaris SRB biofilm, can also be accelerated by injecting magnetite nanoparticles (MNPs) to reach 20 ppm (w / w). Each MNPs dosage costs less than $1. M-MIC does not respond to MNPs or riboflavin

[0045] . Note that MIC (either EET-MIC or M-MIC) can be distinguished from abiotic corrosion using Tafel scan skews and biocide response.

[0203] The apparatus can be used to pre-screen biocide treatment efficacy. The inventors have shown in several journal papers that biocide treatment led to MIC decrease that can be reflected by LPR, EIS and Tafel scans 46], The electrochemical scan data can be used to obtain a biofilm aggressiveness correlation for a specific biocide’s efficacy in an MIC system. MICORP can then be used to predict the KPI (key performance index) of the biocide in future applications using the correlation.

[0204] 2. Use of the apparatus for other applications

[0205] In another application, the apparatus can be used to assess antimicrobial efficacy against biofdms. This includes antimicrobial treatment of environmental biofilms and disease-related biofilms in medicine. Conventional efficacy testing of biofilm treatment in medicine uses a plate with several wells, which means biofilms are grown on Plexiglas. A user can inject microbial broth into the inventors’ biofilm test kit to grow biofilms on the surface of a metal (e.g., 304 stainless steel or carbon steel) that serves as the working electrode. After incubation, an antimicrobial solution can be injected. The antimicrobial can also be added at the beginning of incubation. Electrochemical scans can detect very small corrosion signals. Antimicrobial efficacy can be reflected by jCOrr decrease and Rpincrease. EIS (electrochemical impedance spectrometry) can also be used to provide additional insight into biofilm change. These electrochemical scans can provide near-real-time transient information, and assess the persistence of the antimicrobial kill.

[0206] The apparatus can also be used to screen biocorrosion threats in heat exchangers and other devices in various industries in addition to oil and gas. It can be used to screen different biocides and to decide the proper dosage by testing field water samples’ corrosivity and response to biocide treatment.

[0207] 3. Use of the apparatus to create an online sensor

[0208] FIGS. 27A and 27B show a retractable online biofilm / MIC sensor design with two solid-state electrodes. As described above, in certain embodiments, the electrochemical cell of the apparatus may include a housing that defines an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber when in a first position, and the first and second solid-state electrodes being moveable to a second position positioned outside of the interior chamber. At least one valve may be associated with the housing, wherein the first and second solid-state electrodes are positioned on one side of the valve when in the first position, and wherein the first and second solid-state electrodes are positioned on an opposite side of the valve when in the second position. In another embodiment of the system, there may be a second valve associated with the housing of the apparatus. This second valve is adapted to allow for the introduction of materials into the interior chamber of the electrochemical cell of the apparatus.

[0209] During normal operation, the two electrodes are exposed to the pipeline fluids. Periodically, the electrodes are retracted into the chamber (shown in FIGS. 27 A and 27B as having two valves at its two ends, for example). This interior chamber now becomes an isolated electrochemical cell for injection of electron mediators and biocides. FIG. 28 shows another design utilizing a single full-port valve. It has a cylinder as a shaft that can move up and down for retraction and insertion. The cylinder bottom contains WE and CE / p-RE which are flush with the cylinder’s bottom disc surface. Two electrode wires linking WE and CE / p-RE are embedded in the center of the cylinder itself. The top of the cylinder’ s outer perimeter has threads to seal with the outside housing which is made leak proof with a bonnet. Thus, there is no need for the top valve as in the design in FIG. 27A to create an isolated chamber for injections of electron mediators and biocides. The use of one or two valves allows the sensor electrodes to be exposed to the pipeline fluid to grow biofilms and then be retracted to an isolated chamber for injection of an electron mediator or a biocide for corrosion type diagnosisor biocide efficacy assessment. Existing online sensors such as BioGEORGE (US. Patents 5,246,560 and 5,356,521) do not have a design of an isolated chamber which has been essential for diagnosis of corrosion type and assessment of biocide efficacy assessment using only a small amount of chemical in the chamber.

[0210] When such injections are not needed, the design can simply be a permanently fixed cylinder probe without an outside housing, much like a common commercial ER (electrical resistance) probe for corrosion inside a pipeline. Such a simple probe can still be used to perform LPR, Tafel, EIS and EFM scans to monitor biofilm growth inside the pipeline and biocide efficacy for biofilm treatment. Note that the biocide efficacy study will require a large amount of biocide in the pipeline itself. Our online sensor with a chamber design can test a biocide for efficacy and dosage information using a small amount of biocide in the chamber. This can be done before a large amount of biocide is tested in the pipeline itself. In the chamber- free design, Tafel scan skews can still be used to distinguish MIC from abiotic corrosion. Existing pipelines or other assets that are already fitted with a probe like an ER probe can be retrofitted with the inventors’ electrode choices using the same insertion port on the pipeline wall to monitor biofilm growth, MIC progression and biocide efficacy.[00211J While the present invention can be used for biofilm / MIC detection and biocide treatment, those of ordinary skill in the art will appreciate that the electrochemical scans are also suitable to monitor abiotic corrosion and inhibitor efficacy against abiotic corrosion, because the same electrochemical scans are applicable in both biotic corrosion and abiotic corrosion.

[0212] For an existing pipeline that is already in operation, installation of a sensor can be done without downtime. A commercial hot tap tool can be used to drill a hole while the pipeline is still in operation with gas or liquid flow inside (https: / / www.youtube.com / watch7v-SwDZC 9kPcY). Such a tool is available from Metal Samples Company, 152 Metal Samples Road, Munford, AL 36268). A tubing with two fullport valves is mounted on the hole (FIG. 27A-B). A ball valve is an example of full-port valve. When the valve is fully open, the flow path has the same diameter as the pipe diameter. The ball’s opening may use threads to allow the sensor to move up or down.

[0213] The sensor head has a WE and a CE / p-RE, both solid-state electrodes. Because it is not a miniature device, a separate dedicated RE either having the same conductive material as CE or a different conductive material can be used to have a three-electrode electrochemical cell. All electrodes arc solid-state electrodes to be durable. The sensor head can be deployed into the pipeline flow stream to allow a biofilm to grow on the WE (FIG. 27A-B) so corrosion rate can be measured in near-real time using various electrochemical scans such as LPR, Tafel and EIS scans. Each LPR scan can be as short as 2 min or even 1 min. Other scans take 10X more time. In this deployed mode, the sensor can assess biocide treatment efficacy by comparing corrosion rates before and after biocide treatment in the pipeline or the isolated chamber.

[0214] When riboflavin or another chemical such as magnetite nanoparticles needs to be injected to test whether EET-MIC is present, the sensor can be retracted into the chamber above the bottom valve (FIG. 27B and FIG. 28). By doing so, the injected chemical will not be diluted by the pipeline flow. While the sensor head is inside the isolated chamber, the entire device filled with the actual pipeline liquid becomes an isolated electrochemical cell that can be used to identify corrosion type, and to assess biocide efficacy, in addition to biocide efficacy assessment using the deployed mode in FIG. 27A which requires a large amount of biocide in the pipeline itself.

[0215] The sensor’s electrodes can be fabricated on a flat tip of a sensor rod so when it is deployed inside a pipe, it is flush with the pipe wall, which is a common style in abiotic corrosion probes to make the sensor head less intrusive to pipeline fluid flow. The inventors’ online sensor can be used for devices other than pipelines. They include storage tanks and heat exchangers. It should be noted that the inventors’ standalone biofilm test kit and online sensor can measure corrosion rate for both biocorrosion and abiotic corrosion with the ability to distinguish different types of biocorrosion.

[0216] 4. Biofilm test kit reproducibility

[0217] FIG. 29 is a graph showing D. ferrophilus in two replicate 10 mL biofilm test kit vials each containing an X65 WE and a graphite CE / p-RE in 5 mL EASW. FIG. 29 showsreproducible LPR scans for two replicate test kit vials. Both scans predicted biofilm maturity time at 3 d.

[0218] 5. Effect of vial size for the test kit on electrochemical scan output

[0219] A larger vial contains more culture medium and more planktonic cells. Thus, it potentially builds a biofilm on a WE surface with more sessile cells faster. This leads to higher corrosivity. FIG. 30 proves this hypothesis. It also shows that the general corrosion trend did not change. In particular, FIG. 30 is a graph showing Rpvariations of X65 WEs in 10 mL, 20 mL, and 50 mL bio film test kits (with 5 mL, 10 mL, and 25 mL of EASW, respectively) during 7-d incubation with D. ferrophilus .

[0220] 6. Effect of lower initial planktonic cell concentration

[0221] FIG. 31 is a graph showing Rpvariations of X65 WEs in 10 mL biofilm test kit vials with different initial inoculum dilution rates during 7-d incubation with D. ferrophilus in 5 mL EASW. (Normal inoculum volume: 100:1 for culture medium to inoculum volumetric ratio yielding 106cell / mL initial planktonic cell concentration.) FIG. 31 indicates that when the standard D. ferrophilus inoculum was diluted by 2X and 4X, corrosivity increase in the first 3 days were slowed, but biofilms all matured at 3 d and the differences in corrosivity afterwards were negligible. This means that with the same culture medium nutrient level, fewer planktonic cells initially can slow down biofilm growth, but growth can catch up.

[0222] However, FIG. 32 is a graph showing Rpvariations of X65 WEs in 10 mL biofilm test kit vials with different initial inoculum dilution rates during 7-d incubation with D. ferrophilus in 5 mL EASW. (Normal inoculum volume: 100:1 for culture medium to inoculum volume ratio yielding 106cell / mL initial planktonic cell concentration.) FIG. 32 shows that when the dilution reached 10X, differences in biofilm corrosivity is obvious. At 100X dilution, biofilm did not build up in the first 7 d. This means a much longer time than 7 d will be needed for a sample with very few planktonic cells despite full nutrients being present. Note that if a field sample contains low planktonic cells and at the same time is low in nutrients, it is advisable to inject nutrient chemicals to enhance microbial growth. This can simulateworst-case scenario in the field when contaminants (such as sewer) introduce nutrients that enhance local biofilm growth.

[0223] 7. Portability of the apparatus including electrochemical cell

[0224] The apparatus as described herein is disposable because inexpensive solid-state electrodes can be used in off-shelf serum vials. It is also highly portable for field applications, which is especially useful for medical applications in locations that delicate imaging equipment, dark room (for staining), and chemicals are not readily available for conventional analysis. One application scenario is the use by field medical personnel working in a remote tropical jungle to screen for antibiotic-resistant pathogenic biofilms such as C. difficile biofilm. The potentiostat used for electrochemical scans can be handheld. FIG. 33 shows a portable potentiostat (in particular, it shows a CS100 portable potentiostat from a vendor at https: / / www.corrtest.com.cn / en.). It uses Wi-Fi or Bluetooth connection with an iPhone or Android phone to run electrochemical scan software. It is powered by a rechargeable battery. A specially designed standalone potentiostat instrument can also be used to scan the inventors’ biofilm test kit. It will have push-button keys to select among the aforementioned various electrochemical scan methods. For LPR and Tafel scans, the instrument will directly output Rpand icorr values. An electronic switch will be used to interface the instrument with multiple test kit units with FIG. 34B as an example, so they can be scanned sequentially.

[0225] 8. In vivo applications

[0226] It is possible to embed solid-state electrodes such as a small (e.g., 0.5 cm2) 304 SS thin foil and a small Pt foil on teeth or inside an animal model’s intestine or airway (i.e., respiratory tract). This allows monitoring of biofilm growth and antimicrobial biofilm treatment in situ with near real-time continuous monitoring using electrochemical scans.

[0227] 9. High-throughput microarray electrochemical test kit for multiple samples

[0228] Microarray plates are widely used in testing biological samples. Each plate can have 4, 12, 24, 36, 48 or 96 wells. Each well (see FIG. 34B) may be fitted or printed with twosolid-state electrodes which can be thin films linked with two wires for potentiostat connections. A potentiostat can be used to scan each well (serving as an electrochemical cell) sequentially. A programmable electronic switch can be used to do the switching from one well to the next for connecting to the potentiostat. As a proof of the concept, FIG. 34B shows two wells on a plate (section cut from a 24-well plate) serving as two electrochemical cells. FIG. 35 shows data from an example of using the two wells on a plate design to assess antimicrobial treatment for C. difficile biofilm. In particular, FIG. 35 is a graph showing Rpcurves of 304 SS WEs from LPR measurements during 7-d incubation of C. difficile with and without vancomycin treatment in two wells on a 24-well plate each containing 1.5 mL BHIS medium at 37°C. In summary, FIG. 35 demonstrates that wells from a microplate can be fitted with two solid-state electrodes to assess antibiotic treatment efficacy of a pathogenic biofilm. Because the electrodes are inexpensive, such as plate can be disposable. They are also re-usable if sanitized after each use.

[0229] A microarray plate with electrochemical wells (cells) can be used to test multiple samples (up to 96 samples if a 96-well plate is used). Each well can be used to monitor biofilm growth and antimicrobial treatment for one biofilm sample. Apart from medical applications, such a device can be used for environmental samples such as oil pipeline fluid or sludge samples for high-throughput analysis of biofilm corrosivity and biofilm treatment using antimicrobials.

[0230] EXAMPLE 3

[0231] This example is directed to demonstrating principles of the present invention as they relate to the use of the apparatus, systems, and methods described herein with respect to biofilm and biocorrosive disruption of coatings.

[0232] 1. Introduction

[0233] As described above, coatings are the main method to protect against external pipeline corrosion and other assets including civilian and military assets. Coating damage such as scratches and disbondment can expose the base material to the corrosive environment. Electrochemical methods, especially EIS scans, are routinely used to assess coating damages[36, 37]. The inventors’ coating sensors are based on the same concept used in the inventors’ biofilm / MIC test kit design with two solid-state electrodes (FIG. 36A). If the base material protected by a coating is conductive (e.g., carbon steel pipeline), it can serve as the WE itself without an added WE as shown in FIG. 36B. The sensor can be either portable or permanently fixed to a pipeline or a storage tank location with remote monitoring capability via wireless networking. In particular, FIGS. 36A and 36B are two schematics illustrating field deployment of coating sensors: (FIG. 36A) Pre-deployed separate WE attached to the pipeline coating surface and a temporary CE / p-RE placed above WE or near it before scans, (FIG. 36B) pipeline metal itself serving as WE with a permanently installed CE / p-RE which can have a protective cover opened only during scans to reduce biofouling of its surface.

[0234] Corrosion results in an annual cost of US$4 trillion globally

[0047] . MIC (microbiologically influenced corrosion) is responsible for 20% of all corrosion damages [48,49], It happens in various industrial environments such as underground pipelines, sewage water conduits, recirculating cooling systems, and oil and gas wells [50-54]. Both external and internal MIC are commonly seen in pipelines [55,56]. MIC was also found to be the predominant factor responsible for the perforation and leakage of buried pipelines in soil environments, which are typical examples of external MIC [57,58].

[0235] MIC of metals is the result of interactions between metals, surrounding environments and microorganisms

[0059] . During the soil MIC process, external corrosion of buried pipelines happens when corrosive microorganisms facilitate the electron transport from metals to suitable electron acceptors such as sulfate and nitrate [60,61]. Sulfate reducing bacteria (SRB) play an important role in soil MIC of pipelines since the oxygen level in soil is usually low, which promotes SRB growth

[0062] , They are very aggressive to pipelines as they can directly utilize Fe° as electron donors to harvest energy under the condition of organic carbon starvation [63,64]. Apart from SRB, nitrate reducing bacteria (NRB) is another important group that live in soil. They were also reported to be responsible for severe MIC under anaerobic conditions [65,66]. The wide application of nitrogen fertilizer and biological nitrogen fixation in soil provide NRB a suitable environment to grow [61,67,68]. NRB is also capable of causing MIC when organic carbon source is insufficient

[0069] .

[0236] Coating is an effective approach to prevent corrosion, which offers a physical barrier between the metallic surface and the aggressive environment

[0070] . Different materials such as chromate-based conversion coatings

[0071] , anodized coatings

[0072] , metallic and organic coatings arc widely used to combat corrosion

[0073] . Due to the low cost, versatility and aesthetic attributes, organic coatings have become the single most widely applied method in corrosion protection [74,75]. Besides the barrier effect, organic coatings also have protective mechanisms which offer inhibitive effect, anodic protection and cathodic protection

[0076] . Common organic coatings include organic conversion coatings, thermoplastics, thermosets (e.g., Epoxy resins), elastomers and organic zinc -rich coatings

[0077] .

[0237] However, there are still several factors that limit the application of organic coatings. Some solvent based organic coatings pose a significant environmental issue

[0078] , Besides, multiplied layers of organic coatings usually need to be applied to ensure sufficient corrosion resistance since it is difficult to create a uniform layer without pores

[0079] , Most importantly, the adhesion of the organic coatings to substrate might be poor without appropriate pretreatment

[0080] . The adhesion can also be reduced by water molecules at the interface between metals and organic coatings

[0081] . Coating disbondment is another issue that may lead to coating failures. Blistering, anodic undermining and cathodic disbondment are main causes of coating disbondment

[0082] . When coating failures happen, corrosive species get in contact with metal substrate and will promote corrosion. Therefore, it is important to detect coating failures promptly to stop corrosion development.

[0238] Electrochemical tests are extensively used in MIC studies. Electrochemical techniques such as linear polarization resistance (LPR) and Tafel were found to consistently support weight loss results and reflect corrosion rate trends correctly with additional transient insights [83,84]. Compared with coupon tests, electrochemical tests are easy to perform and can yield near real-time corrosion rates. Tafel scans were used as a tool to effectively distinguish abiotic corrosion from biotic corrosion. Dual-half scans which start from the open circuit potential (OCP) to ±200 mV vs. OCP on the same working electrode (WE) generated similar results as the continuous upward scan (scanning from -200 mV vs. OCP to 200 mV vs. OCP) in abiotic corrosion systems. However, for biotic corrosion, unlike the dual-half scans, continuous upward scans resulted in considerable corrosion potential (ECorr) shifts, and large (e.g., 50%) corrosion current density (z'coir) deviations as well as large distortions includingcompression of the cathodic curve and elongation of the anodic curve. This was because the continuous upward scans initiated with an extreme voltage without any time for the biofilm to adapt. Therefore, Tafel scans can be employed to determine whether biotic corrosion exists.

[0239] X65 carbon steel, a common pipeline steel for oilfield pipelines, and 316L SS(stainless steel) were used to demonstrate coating sensor use. X65 served as WE to simulate pipeline steel. Corrosion-resistance 316L SS served as CE / p-RE. Fully exposed WEs were compared with coated and coating damaged WEs in the sludge to study soil corrosion using electrochemical tests. Tafel scans were performed to determine the corrosion type of soil coiTosion. Scratch tests were conducted to simulate damaged coating conditions on pipelines. The coating disbondment situation was also simulated to investigate whether the present inventors’ online coating sensor could detect this very harmful event.

[0240] 2. Materials and Methods

[0241] 2.1 Sludge, metal, and coating

[0242] Fresh sludge (for soil corrosion) was obtained from an open drainage ditch behind Stocker Center building, Ohio University in Athens, Ohio, USA. The initial pH of the sludge was 6.8 ± 0.2. X65 carbon steel coupons are used in this work. Each exposed X65 carbon steel coupon had an exposed top surface (10 mm x 10 mm square). All other surfaces were protected by inert Epoxy coating (3M Product 323), which is an organic coating that has high adhesion and resistant to acid and alkaline damage in the pH range of 2 - 14

[0085] . Fully coated coupons had all the surfaces painted with inert Epoxy coating. They were used for WE fabrication. Besides, an X65 carbon steel plate (40 mm x 10 mm x 5 mm) was employed to simulate a pipeline’s external surface and it served as a WE in the test. Graphite rods (6.4 mm diameter and 10 mm height) served as the counter electrode / pseudo-reference electrode (CE / p- RE) for the coating and scratch tests. 316L SS coupons (10 mm diameter circle) were used in the coating disbondment test. Elemental compositions of X65 carbon steel and 316L SS are summarized in Table 1.

[0243] Table 1. Elemental compositions (wt. %) of 316L SS and X65 carbon steel (Fe balance).Metal C P S Si Mn Cr Ni Mo V Nb Ti316L 0.02 0.03 0.01 0.38 0.98 16. 11. 1.9SS 2 5 0 2 4 0 4 5X65 0.16 0.02 0.01 0.45 1.659 5 6[00244J 2.2 Exposed WE vs. coated WE

[0245] The corrosion behaviors of exposed and fully coated WEs (FIG. 37) in the aerobic sludge were compared in 450 mL glass bottles containing 200 mL wet aerobic sludge (FIG. 38). (FIG. 37 shows X65 coupons (1 cm2) with different coating conditions used for soil corrosion tests. And FIG. 38 shows a vented 450 mL glass bottle containing electrodes before (left) and after (right) being buried in 200 mL sludge.) A 2-electrode (2E) setup was adopted to conduct electrochemical scans in which the test coupon served as the WE and 316L SS served as both the CE and the p-RE. A PCI4 / 750 potentiostat (Gamry Instruments, Inc., Warminster, PA, USA) was employed to perform LPR and Tafel scans on a daily basis during a 7-d period. LPR was measured from -10 mV vs. OCP to 10 mV vs. OCP with a scan rate of 0.167 mV / s. Tafel curves were obtained using the dual-half scan starting from OCP to - 200 mV and OCP to + 200 mV with a scan rate of 0.167 mV / s on the same working electrode. The performance of the present inventors’ online coating sensor in yielding corrosion rate differences between exposed and coated WEs were evaluated through Rpand icon results from LPR and Tafel scan curves.

[0246] 2.3 Scratching test

[0247] The coupon with coating scratched (with metal surface exposed at scratch lines) as shown in FIG. 37 was put into the same glass cell containing an exposed coupon and a fully coated coupon in the sludge. The scratched coating was used to simulate the coating damage condition. Their corrosion rates were monitored and compared with daily LPR and Tafel scans. In a separate glass bottle, Rpand icon of a fully coated WE was compared with a fully exposed coupon at the first 3 d. Then, at 3 d, the coating was scratched with a knife to generate severalscratching lines to expose metal surface under the lines. The scratched WE was put back into the sludge and its corrosion rates were continuously monitored until 7 d.

[0248] 2.4 Coating disbondment test

[0249] The X65 carbon steel plate was used in the coating disbondment test. Before coating the X65 plate, a plastic tape was applied in a small area (10 mm x 5 mm) on the surface to prevent the direct contact of metal surface and Epoxy coating as indicated in FIG. 39 [FIG. 39 shows a large X65 coupon steel plate WE and 316 SS CE / p-RE used in coating disbondment test, [(the large rectangular box bulge indicates coating disbonded area. The thick vertical center line in the middle of the box indicates location of an incision line to remove the plastic tape in the “pocket” which subsequent allowed sludge and fluid to ingress and come into contact with the bare X65 surface)]. A 316L CE / p-RE was placed on the plate using Epoxy coating underneath the 316L to insulate between the WE and the CE / p-RE. The corrosion rate of the X65 plate was monitored for 3 d using LPR scans. Then, at 3 d, a small 6 mm-long incision was created with a sharp blade to take the tape out, therefore creating a coating disbondment condition between the coating and the bare X65 plate surface. The sludge and fluid were able to move into the gap to cause corrosion. Electrochemical scans were continued to compare the differences in corrosion resistances before and after the coating disbondment.

[0250] 3. Results and Discussion

[0251] FIGS. 40A-B, FIGS. 42A-B, and FIG. 43 show that LPR Rpand Tafel scan icon can be used to detect coating protection condition, with scratched or disbonded coating exhibiting higher corrosivity (lower Rp) caused by the aerobic sludge collected from a drainage ditch behind Stocker Center on Ohio University campus to simulate soil corrosion. X65 served as WE while 316L SS served as CE / p-RE. In particular, FIGS. 40A-B are graphs showing corrosion resistance Rp(FIG. 40A) from LPR measurement, and corrosion current density icon(FIG. 40B) from Tafel scans on coated and exposed X65 WEs in 450 electrochemical glass cell containing 200 mL aerobic sludge over 7-d period. FIGS. 42A-B are graphs showing variations of Rp(FIG. 42A) from LPR measurement, and icon (FIG. 42B) from Tafel scans in scratching test over 7-d period. And FIG. 43 is a graph showing LPR vs. time of X65 in coatingdisbondment test in 450 mL glass bottle containing 200 mL aerobic sludge used to simulate the environment of a buried pipeline exposed to soil.

[0252] 3.1 Coating protection analysis

[0253] In FIG. 40A-B, the initial (i.e., 0-d) Rpvalues of the coated X65 WE and the uncoated WE electrode were similar. During the 7-d period, the Rpcurve of the coated WE remained stable and at a high level. The large Rr, values indicated small or negligible corrosion rates. In contrast, the exposed (uncoated) WE suffered obvious corrosion except for 1 d (biofilm not sufficiently established yet) as reflected by its decreasing 7?ptrend afterwards. The result proved the effectiveness of Epoxy coating in preventing external corrosion. The icon results also suggested the same conclusion. The z'corr values of the coated WE in FIG. 40A-B were consistently low over the 7-d period, while the exposed WE exhibited an increasing trend showing higher corrosion rates. Based on the l / / ?pand z'corr trends, coating decreased corrosion rates by 69% and 53% at 7 d, respectively. These findings proved the feasibility of the online coating sensor in interpreting coating effectiveness in corrosion protection.

[0254] FIG. 40A-B shows. Rp(FIG. 40A) from LPR measurement, and icon (FIG. 40B) from Tafel scans on coated and exposed X65 WEs in 450 electrochemical glass cell containing aerobic sludge over 7-d period.

[0255] FIG. 41 shows the Tafel scan curves obtained with two different methods on the exposed X65 WE in sludge at 3 d. The dual half-scans produced a symmetric Tafel curve. In comparison, the curve from the continuous upward scan had a large compression in the cathodic region (voltage) as well as a considerable elongation in the anodic region. The icon value fitted from the continuous upward scan Tafel curve (0.74 pA / cm2) was 43% lower than that fitted from the dual half-scans ( 1.3 pA / cm2). These results all suggested that X65 corrosion in the sludge belonged to biotic corrosion, rather than abiotic corrosion such as acid corrosion, which is negligible because the sludge’s pH was 6.83 which is circumneutral. Sludges are usually rich in microorganisms of many different types including SRB and NRB.

[0256] 3.2 Scratched coating

[0257] FIGS. 42A-B [which show variations of Rp(FIG. 42A) from LPR measurement, and z'corr (FIG. 42B) from Tafel scans in scratching test over 7-d period] presents Rpand imn variations of WEs in the scratch tests. From 0 d to 3 d, the coated WE and the exposed WE showed similar Rpand icon trends as those in FIGS. 40A-B. At 3 d, the fully coated WE was scratched using a knife to create three 1-cm long cut lines on its coating to expose the base metal underneath. This simulated the situation that coatings on pipelines are occasionally damaged. Within minutes, 7pstarted to decrease for the scratched WE. The corresponding icon also increased and got closer to the exposed WE curve in FIGS. 42A-B. These results indicated that corrosion happened on the partially exposed WE due to the scratches. At 5 d, a largest Rpdecrease of 47% and a largest icon- increase of 41% were observed for the scratched WE compared to the values at 3 d before scratching. In the end at 7 d, RPand icon for the scratched WE recovered to the values before scratching, which could be explained by the fact that only thin lines of metal areas were exposed to sludge, such that the corrosion products on the surface might block the small area, and the passivating effect prevented further corrosion. These results proved the capability of the inventors’ electrochemical online sensor for coating damages.

[0258] Another CE / p-RE material which replaced 316L SS CE / p-RE was used together with X65 WE for the scratching test. The 7-d Rpand z'corr curves in FIGS. 44A-B showed similar results as FIGS. 42A-B where graphite was used as the CE / p-RE. Corrosion rates were low before coating scratching and showing increase after that. This proves the feasibility of different materials serving as the CE / p-RE for the coating sensor. FIG. 44A-B shows variations of Rp(FIG. 44A) from LPR measurement, and z'corr (FIG. 44B) from Tafel scans in scratching test in 450 mL electrochemical glass cell containing X65 WE and 316L SS CE / p-RE buried in 200 mL aerobic sludge over 7-d period.

[0259] 3.3 Coating disbondment

[0260] The coating disbondment test results are shown in FIG. 43. The Rpof the fully coated X65 WE plate stayed at a high level for the first 3 d. After an incision was made with a blade to remove the plastic tape on the metal surface, a disbondment of coating with sludge and fluid ingress was simulated. Minutes after the coating disbondment, an apparent decreasing Rptrend was observed for the following 6 d. The final Rpat 9 d was 69% lower than that at 3 d just before coating disbondment. Based on the 1 / Rptrend, the corrosion rates considerablyincreased due to the coating disbondment. Compared to the previous scratching test, the coating disbondment area was obviously much larger compared to the scratch-damage area. Therefore, a continuously corrosion rate increasing trend was observed without repassivation. This test showed the large consequence of coating disbondment that led to the substrate metal suffering much more increased corrosion. This finding demonstrated the capability of the present inventors’ coating sensor in detecting coating disbondment.

[0261] 4. Coating sensor in future field applications

[0262] In lab tests, the CE / p-RE electrode is clean before each incubation. In the field, if a temporary CE / p-RE is used near a suspected coating damage site just before electrochemical scans, the CE / p-RE is clean. However, a pre-installed CE / p-RE electrode may be covered with deposits / corrosion products and biomass after a long exposure. To prevent CE / p-RE electrode fouling / biofouling, the CE / p-RE electrode needs to be cleaned (e.g., scrapped and brushed) before electrochemical scans. Or, the electrode should be kept clean by using a cover or removable protective coating, etc. for protection while not in scanning. The WE can be a piece of metal made of the same pipeline steel placed at the pipeline coating location that is suspected to have coating damage or is most vulnerable to damage or corrosion (e.g., location submerged in liquid). If desired, a less-corrosion resistant metal can be used as WE for higher corrosion detection sensitivity as early warning. The WE can be the pipeline itself with pre-installed wires in various locations for connection to a potentiostat to detect corrosion at different locations. With such a wire, a spot on the pipeline needs to be exposed for electrical wire connection and sealed with a protective cover before electrochemical scans. FIGS. 36A and 36B explain field deployment scenarios. Wireless communications can be used for online sensors. The same approached using for external pipeline coating damage detection can be used for storage tanks and other assets as long as they are wetted and with possibility for biofilm coverage that leads to corrosion.

[0263] 5. Supplemental information

[0264] The present inventors’ coating sensor working principle was also tested in simulated external pipeline MIC condition using a pure-strain soil microbe. A 10 mL miniatureelectrochemical glass cell (FIG. 45) containing an X65 WE and a 316L SS CE / p-RE in 5 mL Luria-Bertani medium with KNO3 (LB-NO3 medium) was used. The coated WE was compared with the exposed WE in nitrate reducing Pseudomonas aeruginosa MIC. Results in FIG. 46 clearly indicated that compared to the exposed WE, the coated WE had much higher Rpand lower imn, indicating low corrosion rates due to the coating protection. FIG. 45 shows 10 mL electrochemical glass cells containing 5 mL LB-NO3 medium before incubation with nitrate reducing P. aeruginosa to simulate coated metal exposure to an environment with a corrosive microbe. And FIG. 46A-B are graphs showing Rp (FIG. 46A) from LPR measurement, and icon(FIG. 46B) from Tafel scans on coated and exposed (uncoated) X65 WEs in 10 mL electrochemical glass cell containing nitrate reducing P. aeruginosa over 7-d period.

[0265] In the coating sensor design, a thin metal piece may replace large and thick metal pieces as the WE for deployment in multiple locations on a pipeline or storage tank. In the preliminary test shown in FIG. 47A with 316L SS serving as CE / p-REs, the coated thin piece X65 WE (10 mm x 10 mm x 0.4 mm) exhibited stable and high corrosion resistance compared to the exposed one, which proved the MIC protection effect of the coating. The uncoated cold-worked thin X65 WE was compared to the normal X65 WE (10 mm x 10 mm x 1 mm) in FIG. 47B, and the thin WE showed relatively lower Rp, but it reflected the similar Rptrend. Therefore, this cold-worked thin X65 WE was able to reflect the correct corrosion trend as the normal WE without cold work to reduce its thickness. FIG. 47A-B shows Rpvariations of coated and exposed thin (cold-worked) X65 WEs (FIG. 47A) and exposed normal X65 WE vs. thin (uncoated cold-worked) X65 WE (FIG. 47B) in 450 mL electrochemical glass cell containing 200 mL aerobic sludge. It should be noted that a thin metal’s cold work can be removed by the commonly practiced annealing heat treatment.

[0266] 6. Portable biofilm detector / sensor to detect biofilm location

[0267] A portable coating damage sensor can also be used to detect a (hidden) corrosive biofilm on a metal surface such as a storage tank (including aircraft fuel tank) surface and a submerged surface of an asset that is not accessible for direct visual inspection. In FIG. 55, when the RE electrode moves to a certain location (with the base metal as WE) and exhibits abnormally high corrosivity, it means that the location likely has a corrosive biofilm on thesurface. Tafel scan skews can be used to confirm the presence of the biofilm. FIG. 55 is an illustration of biofilm detection on a storage tank using a portable biofilm sensor to pinpoint biofilm location, as long as the biofilm exhibits corrosivity due to biofilm build-up on a bare metal surface or a surface with coating damage.

[0268] EXAMPLE 4

[0269] This example is directed to demonstrating principles of the present invention as they relate to the use of the apparatus, systems, and methods described herein with respect to medical uses, for example, for assessing antibiotic efficacy against biofilms.

[0270] 1. Introduction

[0271] As described above, most microbes in nature live in biofilms rather than as planktonic cells. Biofilms offer an ecological environment that can be very different from the bulk-fluid phase. Biofilms have a sessile cell density easily 100X or 1000X higher per unit volume than planktonic cells in the bulk-fluid phase. This means, underneath a biofilm, the biochemical conditions can be vastly different from those in the bulk-fluid phase. Thus, harmful metabolites can have much higher concentrations underneath a biofilm. For example, the pH (indicator of H+concentration) underneath an acid producing bacterium (APB) biofdm can be far more acidic, which can lead to acid corrosion of metals. Unlike planktonic cells, some sessile cells can harvest electrons from a metal via extracellular electron transfer (EET), causing EET-MIC (microbiologically influenced corrosion) (Li et al., 2018). For pathogenic microbes, a biofilm can be far more virulent than suspended planktonic cells simply because of a locally much higher cell concentration underneath a biofilm.

[0272] Many microbial biofilms are involved in diseases. For example, Pseudomonas aeruginosa biofilm is involved in cystic fibrosis (Hassett et al., 2010). Clostridioides difficile biofilm causes diarrhea and colitis (food-bone diseases) according to US CDC (https: / / www.cdc.gov / cdiff / what-is.html). Biofilms can also grow on the internal walls of catheters used in chemotherapy patients, leading to life-threatening infections because these patients have weakened immune system during chemotherapy (Raad et al., 2007).

[0273] Biofilms arc notoriously recalcitrant toward antimicrobial treatment (Donlan, 2008; Gominet et al., 2017). For example, Pseudomonas aeruginosa infects the airway of cystic fibrosis patients and it is very difficult to treat (Hassett ct ah, 2010). The prevalence of biofilms in chronic wounds was found to be 78.2 % (Malone et ah, 2017). Biofilms are notoriously recalcitrant to treat, often requiring 10X higher dosage of antimicrobials (Hall- Stoodley et al., 2004), because they have various defense mechanisms (Jia et al., 2019).

[0274] Because antibiotics resistance is a serious concern in our modem society nowadays, there is need to accelerate the antibiotic efficacy assessment in vitro and to accelerate the development of new antibiotics or the use of combinations of existing antibiotics. Thus, a high-throughput and convenient way to assess antimicrobial efficacies is highly desirable.

[0275] 2. Difficulties and inaccuracies in conventional cell enumeration

[0276] To enumerate sessile cells in biofilms, sessile cells are scrapped off a solid surface and re-suspended in water or a buffer. Motile bacterial cells can be counted rather accurately using a hemocytometer at 400X magnification because the very small cell dots can be differentiated from artifacts. In antimicrobial treatment studies, a cell suspension’s cell count may be well below the 50,000 cells / mL threshold for accurate hemocytometer counting (of motile bacterial cells) or other imaging methods with stains such as CLSM (confocal laser scanning microcopy) under 400X microscope, because cells are rarely seen in the view field (i.e., not enough of them to be seen). Using 40X magnification can cover a 100X larger view area than 400X magnification, but the bacterial cells are too small to be identifiable even for motile cells or stained cells. MPN (most probably number) serial dilution method can be used to grow cells for counting. CFU (colony forming unit) plating method can also be used to grow colonies for counting. These methods are time-consuming and not accurate. Cell counts in medical biofilm studies often carry wide error bars, making statistical significance hard to achieve in many cases.

[0277] Medical biofilms can be grown on plastic surfaces of plastic plates with multiple wells for cell staining and counting. The color intensity can be used to qualitativelyrepresent biofilm health. Medical biofilms are also grown on stainless steel (SS) such as 304 SS (surgical grade). SS is chosen because carbon steel is not corrosion resistant and thus not- re-usable.

[0278] The inventors found that SS is rather corrosion-resistant against biocorrosion, but a potentiostat can measure minute corrosion signals easily. For example, the inventors can use corrosion current density (icon) from Tafel scans or corrosion resistance (Rp) from linear polarization resistance (LPR), and the combination of charge transfer resistance and film (consisting of biofilm and corrosion product film) resistance (Rct + Rf) in electrochemical impedance spectrometry (EIS) scans. Rp, or Rct + Rf percentage increase, or icon percentage decrease, can be used as antimicrobial inhibition efficiency.

[0279] Biocorrosion is caused by biofilms. Thus, biocorrosion severity can be used to gauge the health of a corrosive biofilm. Corrosion monitoring can be used to monitor different treatment chemicals. Less sessile cells will show less corrosivity. For aerobic biofilms that protect a metal from O2 corrosion, a weakened biofilm will show increased corrosion due to better O2 access to the metal surface. This also can be used to assess antimicrobial efficacy.

[0280] 3. Use of the apparatus of aspects of the present invention

[0281] The present biofilm assessment apparatus, as used for this example, is based on a 10 mL standard serum vial (FIG. 51). [FIG. 51 shows a miniature electrochemical cell based on a 10 mL serum vial with 2 solid-state electrodes: (a) with a vent (a filter on a needle) for aerobic biofilm growth, and (b) without a vent for aerobic biofilm growth]. The vial can be as small as 5 mL or as large as 20 mL, or even larger. The electrochemical cell has two solid-state electrodes. One serves as the working electrode (WE) and another as both counter electrode (CE) and pseudo-reference electrode (p-RE). There is no dedicated RE that requires a liquid electrolyte. This makes the mini cell inexpensive and durable in terms of a long shelf-life. It is used as a biofilm test kit. The WE material can be various types of stainless steel (SS) (e.g., 304 SS, 304L SS, 316 SS, or 316L SS), or carbon steel, or Ti, or a different metal or metal alloy. The CE / p-RE material can be graphite, stainless steel, nickel, chromium or metal alloys, or a different material such as a conductive polymer, a conductivematerial (metal or non-metal) coated with biofouling resistant surface such as CcCh. Each electrochemical cell with 2 solid-state electrodes costs only a few dollars to build in the inventors’ lab. It is disposable. However, after cleaning and sterilization, it becomes reusable. The vial is scaled with a standard rubber septum and an aluminum cap. An inert gas such as N2 can be used in the vial for anaerobic biofilms. For aerobic biofilm growth, a vent port such as a needle with a syringe membrane can be used (FIG. 51a)

[0282] The apparatus is connected to a potentiostat to perform electrochemical scans. Any standard potentiostat with standard corrosion software including EPR, and PDP (potentiodynamic polarization) (also known as Tafel scan) will be sufficient. EIS and EFM (electrochemical frequency modulation) capabilities will offer advanced functions, but they are usually not needed. A standard EPR scan takes only 2 min (or 1 min if a short time is desired). This short scan time can provide near-real time corrosion data which reflect biofilm growth / health. A PDP scan takes 10 times longer. Thus, LPR scans are more convenient.

[0283] By injecting a culture medium with a biofilm-forming microbe sample to start incubation in a mini cell, a biofilm will form on a WE surface and the biofilm’ s electrochemical coiTosion signal can be measured using LPR, PDP, EIS or EFM. Many biofilms exhibit corrosivity even against stainless steels or Ti. The corrosion rates can be extremely small and practically negligible as far as corrosion damage is concerned, but still easily detected using a potentiostat. Some aerobic biofilms can reduce corrosion rates because they form an O2 barrier against O2 corrosion. Either way (causing corrosion or decreasing O2 corrosion), the corrosivity change can be used to characterize the biofilm and antimicrobial efficacy.

[0284] 4. Using the test kit to monitor antimicrobial inhibition of biofilm establishment

[0285] FIG. 48 shows the use of a 10 mL biofilm / MIC test kit to assess 100 ppm (w / w) THPS inhibition of D. ferrophilus biofilm establishment on X65. The uninhibited curve shows that the biofilm increased corrosivity during its growth. It matured at 3 d. After that corrosivity did not increase afterwards. For the inhibited curve, corrosivity did not exhibit any major increase during the 7-d incubation, indicating that a biofilm failed to establish on the X65 WE surface.

[0286] 5. Injections in mixed-culture EET-MIC

[0287] FIG. 49 shows tandem injections of 20 ppm magnetite nanoparticlcs (MNPs) which are known to accelerate EET- MIC

[0026] , followed by 100 ppm THPS, 20 ppm riboflavin electron mediator and another 100 ppm THPS (total 200 ppm in the broth). It shows that MNPs and riboflavin accelerated MIC of X65 carbon steel caused by an oilfield biofilm consortium that contained SRB

[0027] because they both reduced Rp(increased corrosion rate). Thus, like riboflavin electron mediator, MNPs can be used to identify EET-MIC from M-MIC and abiotic coiTosion. FIG. 49 also shows that THPS biocide injections increased Rp(decreased corrosion rate), indicating that THPS was effective against the oilfield biofilm consortium. FIG. 49 also indicate that the 10 mL biofilm test kit can be used to assess biocide efficacy.

[0288] 6. Semi-solid sample testing

[0289] The biofilm / MIC test kit can also be used to analyze a semi-solid sample. FIG. 50 shows LPR response to biofilm growth of a wet sludge sample collected from a drainage ditch behind Stocker Center on Ohio University campus in Athens, Ohio. During the first two days of incubation, corrosivity increased and then reached a peak at 2 d, indicating biofilm maturity at 2 d.

[0290] EXAMPLE 5

[0291] This is another example directed to demonstrating principles of the present invention as they relate to the use of the apparatus, systems, and methods described herein with respect to medical uses, for example, for assessing antibiotic resistance of biofilms and antibiotic efficacy against bio films.

[0292] Some pathogenic microbes form biofilms which make them recalcitrant. A biofilm can utilize several different defense mechanisms [2,31]. Firstly, the EPS in a biofilm presents a mass transfer diffusional barrier for antimicrobial agents to penetrate. Secondly, sessile cells in a biofilm can slow down their metabolism to reduce the intake of antimicrobial agents. Thirdly, a biofilm can form persister cells in the presence of an environmental threat,and these persister cells bounce back to rejuvenate the biofilm once the environmental threat is gone. Fourthly, a biofilm can upregulate the genes that code for antimicrobial resistance. Lastly, sessile cells in a biofilm can utilize efflux pumps to remove toxic chemicals that enter the cells [3].

[0293] To speed up biofilm assessment for antibiotic resistance and better antimicrobial treatment, it is highly desirable to develop a new method to monitor biofilm growth / health continuously with transient information. As long as a pathogenic biofilm shows detectable corrosivity, the biofilm test kit can be used. The biofilm only needs to possess a very small corrosivity, which in the corrosion damage context can be complete negligible. This is because electrochemical corrosion measurements are extremely sensitive.

[0294] Clostridioides difficile is a pathogenic gut bacterium that causes colitis that can lead to death, especially among older patients. It is on the list of USA CDC’s pathogens that require urgent attention. FIGS. 52A and 52B show that although vancomycin took effect (i.e., causing corrosivity to decrease or Rpto increase) slowly in days rather than minutes required for THPS (an industrial biocide and disinfectant) in FIG. 52C, vancomycin increased Rp(i.e., decreased corrosivity) much more, which means vancomycin is more effective although slower acting. FIG. 52D shows that C. difficile biofilm on 304 stainless steel matures (corrosivity levels off) in 3 d in brain heart infusion- supplemented (BHIS) culture medium. FIG. 52C shows that 50 mM metformin (a prescription drug to treat Type 2 diabetes) injection did not exhibit biofilm kill effect (no corrosivity decrease), but 100 ppm THPS biocide yielded a large decrease in a matter of half an hour. C. difficile biofilm establishment was inhibited by 200 mM metformin which was added to the culture medium upon inoculation. The previous 50 mM metformin injection was considered too low because treating a mature biofilm is far more difficult than preventing a biofilm from establishing on a surface.

[0295] In FIGS. 52A-D, C. difficile was incubated for 3 d before injections were made. FIG. 52A shows that vancomycin (an antibiotic) was effective against the biofilm because it reduced the corrosion rate (increased Rp) considerably within 3 hours of its injection. The corrosion rate decreased much more after 24 hours (FIG. 52B). In comparison, metformin (a prescription drug with potential to treat C. difficile) did not have a significant effect against the pre-established biofilm. However, THPS (tetrakis hydroxymethylphosphonium sulfate), a potent industrial biocide, was very effective and much faster acting (< 0.5 hour time to kill) than vancomycin as seen in FIG. 52C. It is more difficult for an antimicrobial to kill a pre-established biofilm than preventing a biofilm from forming on a surface. FIG. 52D shows that metformin successfully prevented C. difficile biofilm formation on 304 SS despite its failure to kill a pre-established biofilm in FIG. 52C. This example shows that the mini cell provided valuable transient antimicrobial efficacy information with convenience, requiring only injections and electrochemical measurements. The time to kill and biofilm prevention duration information would be difficult and less revealing to achieve using conventional testing methods such as microplate incubation. Because the inventors can pre-grow a biofilm and treat the biofilm with an injection of chemical(s), the efficacy reflected by corrosion reduction for the same biofilm is revealing, without suffering data uncertainty (large error bars) in conventional testing that uses two samples: one control biofilm enumerated for sessile cells before antimicrobial treatment and another biofilm after antimicrobial treatment.

[0296] P. aeruginosa is a facultative bacterium that forms biofilms in the airways of cystic fibrosis patients

[0032] , It is also known to be a corrosive microbe both aerobically

[0033] , and also anaerobically as a nitrate reducing bacterium

[0034] . FIG. 53 shows that aerobic P. aeruginosa biofilm reached maturity at 3 d on 304 stainless steel. FIG. 54 shows that biocide treatment of nitrate reducing P. aeruginosa biofilm was successfully assessed using LPR with higher Rpcurves as the result of more effective biocide treatment

[0035] . In FIG. 54, trehalase was used as a biofilm dispersal agent to enhance THPS efficacy. FIG. 53 is a graph showing LPR measurement of 304 SS incubated with aerobic P. aeruginosa in 10 mL biofilm test kit containing 5 mL Luria-Bertani (LB) medium at 37°C. FIG. 54 is a graph showing Rpcurves from LPR measurements during 7-d incubation of galvanized steel with nitrate reducing P. aeruginosa in 10 mL biofilm test kit containing 5 mL LB-NO3 medium at 37°C (modified from

[0035] ). The data in FIG. 54 shows higher corrosion resistance Rpcurve (lower corrosion rate) for better THPS treatment which can be enhanced using trehalase enzyme that regulates biofilm formation.

[0297] The embodiments of the present invention recited herein are intended to be merely exemplary and those skilled in the art will be able to make numerous variations and modifications to it without departing from the spirit of the present invention. Notwithstandingthe above, certain variations and modifications, while producing less than optimal results, may still produce satisfactory results. All such variations and modifications are intended to be within the scope of the present invention as defined by the claims appended hereto.

[0298] INCORPORATION BY REFERENCE

[0299] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

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Claims

CLAIMS:What is claimed is:

1. An apparatus for the detection of biofilms and biocorrosion, the apparatus comprising: an electrochemical cell including first and second solid-state electrodes; wherein the first solid-state electrode is a working electrode and the second solid- state electrode is a counter electrode; wherein the electrochemical cell does not include a dedicated reference electrode in addition to the first and second solid-state electrodes.

2. The apparatus of claim 1, the electrochemical cell further comprising a housing and a closure that define an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber, and wherein the closure is adapted to provide access to the interior chamber.

3. The apparatus of claim 1 or claim 2, wherein the first solid-state electrode comprises a conductive metal.

4. The apparatus of claim 3, wherein the first solid-state electrode comprises a conductive metal chosen from iron, carbon steel, stainless steel, galvanized steel, copper, zinc, aluminum, galvanized steel, bronze, brass, titanium, chromium steel, and nickel.

5. The apparatus of any of claims 1-4, wherein the second solid-state electrode comprises a material chosen from graphite, a conductive metal, a conductive polymer, a conductive organic material, a conductive inorganic material, a coated metal, and a coated polymer.

6. The apparatus of claim 5, wherein the material is a conductive metal, and the conductive metal is chosen from copper, platinum, tantalum, nickel, molybdenum, chromium, steel, stainless steel, and brass.

7. The apparatus of claim 5, wherein the material includesplastic or glass.

8. The apparatus of any of claims 5-7, wherein the material is a coated metal, coated ceramic, or coated polymer, wherein the coating is conductive or semi-conductive.

9. The apparatus of claim 8, wherein the metal or polymer is coated with cerium oxide, platinum, copper, tantalum, nickel, molybdenum, chromium, steel, stainless steel, or brass.

10. The apparatus of claim 5, wherein the second solid-state electrode operates as both a counter electrode and a pseudo-reference electrode.

11. The apparatus of claim 2, wherein the interior chamber has a volume, and wherein the volume ranges from 1 mL to 200 mL12. The apparatus of claim 11, wherein the volume is chosen from 5 mL, 7mL, 10 mL, 15 mL, 20 mL, 30 mL, 50 mL, 75 mL, 100 mL, 125 mL, and 200 mL.

13. The apparatus of claim 2, wherein the interior chamber contains nitrogen gas, argon gas, or air.

14. The apparatus of claim 2, wherein the closure is a septum.

15. The apparatus of claim 1, further comprising a potentiostat operatively connected to the electrochemical cell.

16. The apparatus of claim 1, the electrochemical cell further comprising a housing that defines an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber.

17. The apparatus of claim 16, wherein the housing comprises a well of a microplate.

18. The apparatus of claim 17, wherein the first and second solid-state electrodes are printed or attached on a surface of the well, such that the space defined by a wall of the well defines the interior chamber of the electrochemical cell.

9. An online biofilm sensor for the detection of biofilms and biocorrosion, the apparatus comprising: an electrochemical cell including a housing and a closure that define an interior chamber; first and second solid-state electrodes positioned in the interior chamber, wherein the first solid-state electrode is a working electrode and the second solid-state electrode is a counter electrode; and a dedicated reference electrode in addition to the first and second solid-state electrodes.

20. A system for the detection of biofilms and biocorrosion, for the assessment of biofilms and biocorrosion, and for the assessment of treatment of same, the system comprising: a component that is potentially subject to biocorrosion, biofouling or to biofilms associating therewith, and an apparatus as recited in claim 1.

21. The system of claim 20, wherein the component includes an interior space adapted to contain at least one liquid therein.

22. The system of claim 21, wherein the component is a conduit for transport of the at least one liquid.

23. The system of claim 22, wherein the conduit is a pipeline.

24. The system of claim 21, wherein the component has an interior surface, at least a portion of which contacts the at least one liquid, when the at least one liquid is present.

25. The system of claim 24, wherein the interior surface of the component comprises a material.

26. The system of claim 25, wherein the first solid-state electrode comprises the same material as the interior surface of the component.

27. The system of claim 26, wherein the material is a conductive metal.

28. The system of claim 27, wherein the conductive metal is chosen from iron, carbon steel, stainless steel, copper, zinc, aluminum, galvanized steel, bronze, brass, titanium, chromium steel, and nickel.

29. The system of claim 21 , the electrochemical cell further comprising a housing that defines an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber when in a first position, and the first and second solid-state electrodesbeing moveable to a second position positioned outside of the interior chamber of the apparatus and inside the interior space of the component.

30. The system of claim 29, further comprising a valve associated with the housing, wherein the first and second solid-state electrodes are positioned on one side of the valve when in the first position, and wherein the first and second solid-state electrodes are positioned on an opposite side of the valve when in the second position.

31. The system of claim 30, further comprising a second valve associated with the housing, the second valve capable of allowing for the introduction of materials into the interior chamber of the electrochemical cell.

32. The system of claim 20, wherein the component includes an exterior surface.

33. The system of claim 31, wherein the first solid-state electrode is placed on and in contact with the exterior surface of the component.

34. The system of claim 33, wherein the second solid-state electrode is placed near’ the exterior surface of the component.

35. The system of claim 33, wherein the second solid-state electrode is placed on and in contact with the exterior surface of the component.

36. A system for the detection of biofilms and biocorrosion, for the assessment of biofilms, biocorrosion and biofouling, and for the assessment of treatment of same, the system comprising: a conductive component that is potentially subject to biocorrosion or to biofilms associating therewith wherein the component comprises a metal, and a solid-state electrode; wherein a surface of the component is used as a working electrode, and wherein the solid-state electrode is used as a counter electrode, such that the surface of the component and the solid-state electrode comprise an electrochemical cell.

37. The system of claim 36, wherein the electrochemical cell further comprises a liquid in contact with the working electrode and the solid-state electrode.

38. The system of claim 36, wherein the component includes an interior space adapted to contain at least one fluid therein.

39. The system of claim 38, wherein the component is a conduit for transport of the at least one liquid.

40. The system of claim 39, wherein the conduit is a pipeline or a storage tank.

41. The system of claim 36, wherein the component is a metal pillar exposed to a liquid including water or a liquid suspension of particles including water.

42. The system of claim 36, wherein the solid-state electrode comprises a material chosen from graphite, a conductive metal, a conductive polymer, a conductive organic material, a conductive inorganic material, a coated metal, and a coated polymer.

43. The apparatus of claim 42, wherein the material is a conductive metal, and the conductive metal is chosen from stainless steel, copper, platinum, tantalum, nickel, molybdenum, chromium, steel, and brass.

44. The apparatus of claim 42, wherein the material is a plastic, ceramic or glass.

45. The apparatus of any of claims 42-44, wherein the material is a coated metal, ceramic or coated polymer, wherein the metal, ceramic or polymer is coated with cerium oxide or platinum.

46. A method for detecting biofilms and biocorrosion, for distinguishing types of corrosion, or for assessing antimicrobial treatment of biofilms and biocorrosion, the method comprising: obtaining a sample from a component having at least one surface subject to corrosion; introducing the sample to an apparatus including an electrochemical cell comprising first and second solid-state electrodes, wherein the first solid-state electrode is a working electrode and the second solid-state electrode is a counter electrode; wherein the electrochemical cell does not include a dedicated reference electrode in addition to the first and second solid-state electrodes; and testing the working electrode to determine if a biofilm has grown on the working electrode.

47. The method of claim 46, wherein the sample is a liquid or a liquid suspension of particles.

48. The method of claim 46, wherein the component has a defined interior space, and the sample is obtained from the interior space of the component.

49. The method of claim 3, wherein the at least one surface of the component is a surface that faces the interior space of the component, such that any sample in the interior space is capable of contacting the at least one surface.

50. The method of claim 3, wherein the component is a conduit for transport of the at least one fluid.

51. The method of claim 3, wherein the component is a pipeline, a storage tank, a metal wall or a metal pillar exposed to a fluid containing water.

52. The method of claim 1, wherein the apparatus further comprises a housing and a closure that define an interior chamber, with the first and second solid-state electrodes being positioned in the interior chamber, and wherein the closure is adapted to provide access to the interior chamber.

53. The method of claim 7, wherein the interior chamber has a volume, and wherein the volume ranges from 1 mL to 200 mL.

54. The method of claim 7, wherein the closure has a septum, and wherein introducing the sample to the apparatus comprises injecting the sample through the septum of the apparatus.

55. The method of claim 48, wherein introducing the sample comprises moving at least the two solid-state electrodes from the interior chamber of the apparatus into the interior space of the component.

56. The method of claim 55, further comprising subsequently moving the solid-state electrodes from the interior space of the component back into the interior chamber of the apparatus.

57. The method of claim 56, wherein the apparatus and component are adjacent one another, and further comprising a valve that allows for communication between the interior chamber of the apparatus and the interior space of the component, wherein moving the solid-state electrodes comprises opening the valve to allow the solid-state electrodes to pass from the interior chamber to the interior space, or to retract from the interior space to the interior chamber.

58. The method of claim 48, wherein introducing the sample comprises causing at least a portion of the sample to move from the interior space of the component to the interior chamber of the apparatus.

59. The method of claim 58, wherein the apparatus and component are adjacent one another, and further comprising a valve that allows for communication between the interior chamber of the apparatus and the interior space of the component, and wherein opening the valve allows bulk fluid and the solid-state electrodes to move from the interior space to the interior chamber.

60. The method of claim 1, wherein the at least one surface is an external surface of the component, and wherein at least a portion of the external surface is used as the working electrode.

61. The method of claim 1, further comprising incubating the sample and working electrode prior to testing the working electrode.

62. The method of claim 1, wherein testing the working electrode further comprises performing an electrochemical scan chosen from open circuit potential, linear polarization resistance, potentiodynamic polarization, electrochemical impedance spectroscopy, and electrochemical frequency modulation.

63. The method of claim 17, further comprising a portable or bench-top potentiostat operatively connected to the apparatus, and wherein the potentiostat is used in the electrochemical scan.

64. The method of claim 17, further comprising determining corrosion rate or corrosion rate trend from the results of the scan.

65. The method of claim 64, further comprising correlating biofilm growth or efficacy of a biocide from the corrosion rate, from the inverse of corrosion resistance), or from the corrosion rate trend.

66. The method of claim 46, further comprising introducing a biocide and an electron mediator into the interior chamber of the apparatus.

67. The method of claim 66, wherein the electron mediator is introduced before introduction of the biocide.

68. The method of claim 66, wherein the biocide is tetrakis-hydroxymethyl phosphonium sulfate (THPS), glutaraldehyde, alkyldimethylbenzylammonium chloride (ADBAC), 2,2- dibromo-3-nitrilopropionamide (DBNPA), or anthraquinone.

69. The method of claim 66, wherein the biocide is an antibiotic, and the antibiotic is chosen from vancomycin, amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin,metronidazole, azithromycin, sulfamethoxazole, trimethoprim, clavulanate, and levofloxacin.

70. The method of claim 67, where in the electron mediator is riboflavin (or other flavins), and magnetite nanoparticles.

71. The method of claim 66, wherein testing the working electrode further comprises performing at least one potentiodynamic polarization scan.

72. The method of claim 24, further comprising determining whether any corrosion is biotic corrosion or abiotic corrosion, or determining type of microbiologically induced corrosion, based on the results of at least one potentiodynamic polarization scan.

73. A method for detecting biofilms or assessing antimicrobial treatment of biofilms, in vivo, the method comprising: inserting first and second solid-state electrodes into the intestines or respiratory tract of a mammal, wherein the first solid-state electrode is a working electrode and the second solid-state electrode is a counter electrode; and testing the working electrode to determine if a biofilm has grown on the working electrode, or to assess treatment of a biofilm.

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