Sessile Cell Count Research Articles

Preliminary Investigation of Utilization of a Cellulose-Based Polymer in Enhanced Oil Recovery by Oilfield Anaerobic Microbes and its Impact on Carbon Steel Corrosion

Water injection increases reservoir pressure in enhanced oil recovery (EOR). Among other oilfield performance chemicals, an EOR polymer is added to the injection water to provide the viscosity necessary for effective displacement of viscous crude oil from the reservoir formation. However, these organic macromolecules may be degraded by microbes downhole, causing undesirable viscosity loss. The organic carbon utilization by the microbes promotes microbial metabolism, thus potentially exacerbating microbiologically influenced corrosion (MIC). In this preliminary laboratory investigation, 3,000 ppm (w/w) carboxymethyl cellulose sodium (CMCS), a commonly used EOR polymer, was found to be utilized by an oilfield biofilm consortium. This oilfield biofilm consortium consisted of bacteria (including that can degrade large organic molecules), sulfate-reducing bacteria (SRB), and other microorganisms. A 30-day incubation in 125 mL anaerobic vials was conducted with an artificial seawater medium without yeast extract and lactate supplements at 37°C. The polymer biodegradation led to 16% viscosity loss in the broth and a 30× higher SRB sessile cell count. Slightly increased MIC weight loss and pitting corrosion were observed on C1018 carbon steel coupons. Thus, the use of CMCS in EOR should take into the consideration of microbial degradation and its impact on MIC.

Corrosion of Cu by a sulfate reducing bacterium in anaerobic vials with different headspace volumes

Microbiologically influenced corrosion (MIC) of copper by Desulfovibrio vulgaris, a sulfate reducing bacterium (SRB), was investigated in anaerobic vials with a fixed broth volume of 40 mL but varied headspace volumes (10 mL, 85 mL 160 mL). It was found that the headspace volume variation had a very large effect on the dissolved [H2S] in the broth and the cell counts of planktonic and sessile cells, as well as Cu corrosion severity. A 16× smaller headspace led to a 1.6-fold increase in the dissolved [H2S], a 13-fold decrease in sessile SRB cell count, a 32-fold decrease in planktonic cell count and a 3.7-fold increase of Cu weight loss. SEM images revealed that different headspace volumes caused different corrosion patterns on the immersed coupons. With a lower headspace volume, pitting corrosion was observed, while with a higher headspace volume, intergranular corrosion was seen. The results confirmed that SRB MIC of Cu belongs to metabolite-MIC (M-MIC) by H2S, unlike SRB MIC of carbon steel that belongs to extracellular electron transfer-MIC (EET-MIC) that is directly correlated with sessile cell counts rather than dissolved [H2S]..

Effects of ferrous ion concentration on microbiologically influenced corrosion of carbon steel by sulfate reducing bacterium Desulfovibrio vulgaris

Ferrous ion (Fe2+) in a sulfate reducing bacteria (SRB) culture medium is known to enhance microbiologically influenced corrosion (MIC) of carbon steel, but the underlining mechanism is controversial. This work showed that it was due to better sessile cell growth that was likely attributed to Fe2+ detoxification of H2S. Two hundred ppm (w/w) initial Fe2+ in the ATCC 1249 medium achieved a 4.7 times higher Desulfovibrio vulgaris sessile cell count and 5.0 times higher C1018 carbon steel weight loss, compared to 20 ppm. Linear polarization resistance, electrochemical impedance spectroscopy and potentiodynamic polarization measurements corroborated weight loss and pitting data trends.

Effects of d-Phenylalanine as a biocide enhancer of THPS against the microbiologically influenced corrosion of C1018 carbon steel

Microbiologically influenced corrosion (MIC) is caused by biofilms such as those of sulfate reducing bacteria (SRB). To mitigate MIC, biocide treatment is often needed. Tetrakis (hydroxymethyl) phosphonium sulfate (THPS) is an environmentally friendly biocide that is often used in the oil and gas industry. However, its prolonged use leads to biocide resistance, leading to dosage escalation. A biocide enhancer can be used to slow down the trend. In recent years, d-amino acids have been investigated as an enhancer for THPS and other biocides. Published works used anaerobic vials and flow devices, which could not reveal the real-time changes of the biocide treatment on corrosion. In this work, it was proven that the biocide enhancement effects of d-Phenylalanine (d-Phe) on THPS against the Desulfovibrio vulgaris biofilm on C1018 carbon steels could be assessed in real time using linear polarization resistance and electrochemical impedance spectroscopy to collaborate sessile cell count, weight loss and pitting depth data. The results showed that 500 ppm (w/w) d-Phe effectively enhanced 80 ppm THPS against MIC by the D. vulgaris (a corrosive SRB) biofilm. The sessile cell count and pit depth were all reduced with the enhancement of d-Phe.

Antimicrobial Cu-bearing 2205 duplex stainless steel against MIC by nitrate reducing Pseudomonas aeruginosa biofilm

In industrial and clinical settings, microbiologically influenced corrosion (MIC), also known as biocorrosion, is a major problem associated with materials degradation and infection. To mitigate biofilms, a Cu-bearing 2205 duplex stainless steel (2205-Cu DSS) was created by researchers to utilize the antimicrobial ability of copper. In this study, nitrate reducing Pseudomonas aeruginosa biofilm was grown as a nitrate reducing bacterium to investigate the antimicrobial efficacy and the MIC inhibition efficacy of 2205-Cu DSS under anaerobic condition. The results showed that both biofilm sessile cell count and electrochemically-measured corrosion rate were reduced compared with the 2205 duplex stainless steel (2205 DSS) control.

Anaerobic Corrosion of 304 Stainless Steel Caused by the Pseudomonas aeruginosa Biofilm.

Pseudomonas aeruginosa is a ubiquitous bacterium capable of forming problematic biofilms in many environments. They cause biocorrosion of medical implants and industrial equipment and infrastructure. Aerobic corrosion of P. aeruginosa against stainless steels has been reported by some researchers while there is a lack of reports on anaerobic P. aeruginosa corrosion in the literature. In this work, the corrosion by a wild-type P. aeruginosa (strain PAO1) biofilm against 304 stainless steel (304 SS) was investigated under strictly anaerobic condition for up to 14 days. The anaerobic corrosion of 304 SS by P. aeruginosa was reported for the first time. Results showed that the average sessile cell counts on 304 SS coupons after 7- and 14-day incubations were 4.8 × 107 and 6.2 × 107 cells/cm2, respectively. Scanning electron microscopy and confocal laser scanning microscopy corroborated the sessile cell counts. The X-ray diffraction analysis identified the corrosion product as iron nitride, confirming that the corrosion was caused by the nitrate reducing biofilm. The largest pit depths on 304 SS surfaces after the 7- and 14-day incubations with P. aeruginosa were 3.9 and 7.4 μm, respectively. Electrochemical tests corroborated the pitting data.

Effects of biogenic H2S on the microbiologically influenced corrosion of C1018 carbon steel by sulfate reducing Desulfovibrio vulgaris biofilm

The role of biogenic H2S in the microbiologically influenced corrosion (MIC) of carbon steel was investigated. Desulfovibrio vulgaris (ATCC 7757), a sulfate reducing bacterium, was tested against C1018 carbon steel in anaerobic vials with three different sizes, each filled with 40mL of ATCC 1249 culture medium, providing headspace volumes of 10mL, 85mL and 160mL, respectively for H2S to escape. Results showed that a larger headspace led to a lower H2S concentration in the culture medium, and this increased the sessile cell count and made the iron sulfide film thinner, resulting in increased MIC.

Laboratory testing of enhanced biocide mitigation of an oilfield biofilm and its microbiologically influenced corrosion of carbon steel in the presence of oilfield chemicals

Microbiologically influenced corrosion (MIC) is prevalent in the oil and gas industry. Problematic biofilms cause MIC and reservoir souring. A high biocide concentration is usually required to mitigate biofilms compared with planktonic cells. This causes economic and environmental concerns. A biocide enhancer can make a biocide more effective using the same or lower biocide dosage. In this work, an equimolar mixture of 100 ppm (w/w) of four D-amino acids (D-methionine, D-tyrosine, D-tryptophan, and D-leucine) labeled as D-mix enhanced 100 ppm tetrakis (hydroxymethyl) phosphonium sulfate (THPS) against a field biofilm consortium on C1018 carbon steel coupons. In order to test chemical compatibilities, D-amino acids were added together with THPS and enhanced oil recovery chemicals (a polymer, a surfactant, a corrosion inhibitor, and a scale inhibitor) to treat the mature biofilm consortium. After a 7-day biofilm removal test in 125 ml anaerobic vials, the cocktail of 100 ppm THPS +100 ppm D-mix achieved extra logs of reduction in sessile cell counts compared with the 100 ppm THPS alone treatment. The combination also achieved lower weight loss and smaller maximum pit depths. Electrochemical tests corroborated the weight loss and pitting data.

Enhanced Biocide Treatments with D-amino Acid Mixtures against a Biofilm Consortium from a Water Cooling Tower.

Different species of microbes form mixed-culture biofilms in cooling water systems. They cause microbiologically influenced corrosion (MIC) and biofouling, leading to increased operational and maintenance costs. In this work, two D-amino acid mixtures were found to enhance two non-oxidizing biocides [tetrakis hydroxymethyl phosphonium sulfate (THPS) and NALCO 7330 (isothiazoline derivatives)] and one oxidizing biocide [bleach (NaClO)] against a biofilm consortium from a water cooling tower in lab tests. Fifty ppm (w/w) of an equimass mixture of D-methionine, D-leucine, D-tyrosine, D-tryptophan, D-serine, D-threonine, D-phenylalanine, and D-valine (D8) enhanced 15 ppm THPS and 15 ppm NALCO 7330 with similar efficacies achieved by the 30 ppm THPS alone treatment and the 30 ppm NALCO 7330 alone treatment, respectively in the single-batch 3-h biofilm removal test. A sequential treatment method was used to enhance bleach because D-amino acids react with bleach. After a 4-h biofilm removal test, the sequential treatment of 5 ppm bleach followed by 50 ppm D8 achieved extra 1-log reduction in sessile cell counts of acid producing bacteria, sulfate reducing bacteria, and general heterotrophic bacteria compared with the 5 ppm bleach alone treatment. The 10 ppm bleach alone treatment showed a similar efficacy with the sequential treatment of 5 ppm bleach followed by 50 ppm D8. The efficacy of D8 was found better than that of D4 (an equimass mixture of D-methionine, D-leucine, D-tyrosine, and D-tryptophan) in the enhancement of the three individual biocides against the biofilm consortium.

Mitigation of a nitrate reducing Pseudomonas aeruginosa biofilm and anaerobic biocorrosion using ciprofloxacin enhanced by D-tyrosine

Pseudomonas aeruginosa (PA) is a ubiquitous microbe. It can form recalcitrant biofilms in clinical and industrial settings. PA biofilms cause infections in patients. They also cause biocorrosion of medical implants. In this work, D-tyrosine (D-tyr) was investigated as an antimicrobial enhancer for ciprofloxacin (CIP) against a wild-type PA biofilm (strain PAO1) on C1018 carbon steel in a strictly anaerobic condition. Seven-day biofilm prevention test results demonstrated that 2 ppm (w/w) D-tyr enhanced 30 ppm CIP by achieving extra 2-log sessile cell reduction compared with the 30 ppm CIP alone treatment. The cocktail of 30 ppm CIP + 2 ppm D-tyr achieved similar efficacy as the 80 ppm CIP alone treatment in the biofilm prevention test. Results also indicated that the enhanced antimicrobial treatment reduced weight loss and pitting corrosion. In the 3-hour biofilm removal test, the cocktail of 80 ppm CIP + 5 ppm D-tyr achieved extra 1.5-log reduction in sessile cell count compared with the 80 ppm CIP alone treatment. The cocktail of 80 ppm CIP + 5 ppm D-tyr achieved better efficacy than the 150 ppm CIP alone treatment in the biofilm removal test.

Electrochemical Testing of Biocide Enhancement by a Mixture ofd-Amino Acids for the Prevention of a Corrosive Biofilm Consortium on Carbon Steel

Problematic biofilms cause microbiologically influenced corrosion (MIC) and biofouling in many industries such as the oil and gas industry, water utilities, and the power generation industry. Equipment failures cause not only economic losses but also environmental damages. A high biocide concentration is required to treat mixed-culture biofilms due to their various defense mechanisms. A biocide enhancer can reduce the dosage or make a biocide more effective. In this work, an equimolar mixture with 100 ppm (w/w) of four d-amino acids (d-tyrosine, d-methionine, d-leucine, and d-tryptophan) labeled as d-mix enhanced 60 ppm alkyldimethylbenzylammonium chloride (ADBAC) against a field biofilm consortium on C1018 carbon steel coupons by achieving at least an extra 2-log reduction of sessile cell counts in the biofilm prevention test. Scanning electron microscopy images, confocal laser scanning microscopy images, weight loss, and pitting data all corroborated the electrochemical tests.

Mitigation of the Desulfovibrio vulgaris biofilm using alkyldimethylbenzylammonium chloride enhanced by d-amino acids

d-tyrosine and d-methionine that are naturally occurring chemicals were investigated as enhancers for alkyldimethylbenzylammonium chloride (ADBAC) against the Desulfovibrio vulgaris biofilm on C1018 coupons surfaces in ATCC 1249 culture medium. The 7-day biofilm prevention results showed that the d-amino acids alone had only limited effects on SRB sessile cell counts. Ten ppm (w/w) ADBAC alone achieved a 1-log reduction in SRB sessile cell count compared with the untreated control. The combination of 1 ppm d-tyrosine + 10 ppm ADBAC achieved 3 extra log reduction and 50 ppm d-methionine + 10 ppm ADBAC achieved 2 extra log reduction compared with the 10 ppm ADBAC alone treatment. In the biofilm removal test, both combinations exhibited 2 extra log reduction compared with the 10 ppm ADBAC alone treatment. These results were supported by scanning electron microscope images and confocal laser scanning microscope images of the biofilms.

Effects of oregano essential oil and carvacrol on biofilms of Staphylococcus aureus from food-contact surfaces

This study characterized the biofilm-forming ability of Staphylococcus aureus from food-contact surfaces and evaluated the effects of the essential oil from Origanum vulgare L. (oregano; OVEO) and carvacrol (CAR) against the planktonic and sessile cells of selected isolates, as well as their effects on biofilm formation on stainless steel over time. In microtiter plate (MtP) assay, 97.2% (70/72) of the isolates were considered biofilm-producers, while in assays using Congo Red agar, 88.9% (64/72) of the isolates presented biofilm-forming ability. The detection of icaA, icaB, icaC and icaD genes varied among the 72 S. aureus isolates; however, all isolates (n = 39) classified as strong biofilm producers in Mtp assay were positive for icaA and icaD. For eight out the ten tested isolates, the minimum inhibitory concentration (MIC) of OVEO and CAR was one-fold higher against sessile cells than planktonic cells. MICs against sessile cells were effective to eradicate pre-formed biofilms on polystyrene. Sub-MIC doses of 2.5 and 1.25 μL/mL of OVEO and 1.25, 0.62 and 0.31 μL/mL of CAR in meat broth decreased the sessile cell counts of isolates on stainless steel surfaces over time (360 h). These effects varied with the OVEO and CAR concentrations and exposure times. For all tested isolates, an increase in sessile cell counts was observed after 288 h exposure to 0.625 μL/mL of OVEO. The results showed high incidence of biofilm producers among S. aureus isolated from food-contact surfaces and efficacy of OVEO and CAR to inhibit planktonic and sessile cells. The concentrations of these antimicrobials used to control S. aureus biofilms should be cautiously considered because of possible inductive biofilm production effects.

An investigation of the antibacterial ability and cytotoxicity of a novel cu-bearing 317L stainless steel.

In order to solve the challenging problem of microbial infections caused by microorganisms on medical implants, it is imperative to develop novel antimicrobial biomaterials. This work demonstrated that 317L-Cu stainless steel (SS), created by adding copper through a solution and aging heat treatment process, exhibited good antibacterial properties against staphylococcus aureus, achieving 2 log reduction of planktonic cells after 5 days of incubation. In this study, the antibacterial test was performed using the plate count method, the fluorescence cell staining method and the quantitative polymerase chain reaction (qPCR) method. It is well known that a high concentration of copper ion can lead to cytotoxicity. This work explored the cytotoxicity of 317L-Cu SS through real-time cell analysis (RTCA). Experimental results demonstrated that the 317L-Cu SS possessed a satisfactory antibacterial ability against S. aureus, and the antibacterial rate based on the reduction of sessile cell count reached 98.3% after 24-hour treatment. The bacterial adhesion and the biofilm thickness were considerably reduced by the 317L-Cu SS. The results of RTCA suggested that 317L-Cu SS did not introduce cytotoxicity to mouse cells, indicating its suitability as a medical implant material.

Glyceryl trinitrate and caprylic acid for the mitigation of the Desulfovibrio vulgaris biofilm on C1018 carbon steel.

Microbiologically influenced corrosion (MIC), also known as biocorrosion, is caused by corrosive biofilms. MIC is a growing problem, especially in the oil and gas industry. Among various corrosive microbes, sulfate reducing bacteria (SRB) are often the leading culprit. Biofilm mitigation is the key to MIC mitigation. Biocide applications against biofilms promote resistance over time. Thus, it is imperative to develop new biodegradable and cost-effective biocides for large-scale field applications. Using the corrosive Desulfovibrio vulgaris (an SRB) biofilm as a model biofilm, this work demonstrated that a cocktail of glyceryl trinitrate (GTN) and caprylic acid (CA) was very effective for biofilm prevention and mitigation of established biofilms on C1018 carbon steel coupons. The most probable number sessile cell count data and confocal laser scanning microscope biofilm images proved that the biocide cocktail of 25 ppm (w/w) GTN + 0.1% (w/w) CA successfully prevented the D. vulgaris biofilm establishment on C1018 carbon steel coupons while 100 ppm GTN + 0.1% CA effectively mitigated pre-established D. vulgaris biofilms on C1018 carbon steel coupons. In both cases, the cocktails were able to reduce the sessile cell count from 10(6) cells/cm(2) to an undetectable level.

Effective removal of staphylococcal biofilms by the endolysin LysH5.

Staphylococcal biofilms are a major concern in both clinical and food settings because they are an important source of contamination. The efficacy of established cleaning procedures is often hindered due to the ability of some antimicrobial compounds to induce biofilm formation, and to the presence of persister cells, a small bacterial subpopulation that exhibits multidrug tolerance. Phage lytic enzymes have demonstrated antimicrobial activity against planktonic and sessile bacteria. However, their ability to lyse and/or select persister cells remains largely unexplored so far. In this work, the lytic activity of the endolysin LysH5 against Staphylococcus aureus and Staphylococcus epidermidis biofilms was confirmed. LysH5 reduced staphylococcal sessile cell counts by 1–3 log units, compared with the untreated control, and sub-inhibitory concentrations of this protein did not induce biofilm formation. LysH5-surviving cells were not resistant to the lytic activity of this protein, suggesting that no persister cells were selected. Moreover, to prove the lytic ability of LysH5 against this subpopulation, both S. aureus exponential cultures and persister cells obtained after treatment with rifampicin and ciprofloxacin were subsequently treated with LysH5. The results demonstrated that besides the notable activity of endolysin LysH5 against staphylococcal biofilms, persister cells were also inhibited, which raises new opportunities as an adjuvant for some antibiotics.

Attachment of Listeria innocua to Polystyrene: Effects of Ionic Strength and Conditioning Films from Culture Media and Milk Proteins

It is recognized that bacterial adhesion usually occurs on conditioning films made of organic macromolecules absorbed to abiotic surfaces. The objectives of this study were to determine the extent to which milk protein-coated polystyrene (PS) pegs interfere with biofilm formation and the synergistic effect of this conditioning and hypertonic growth media on the bacterial adhesion and biofilm formation of Listeria innocua, used as a nonpathogenic surrogate for Listeria monocytogenes. PS pegs were uncoated (bare PS) or individually coated with whey proteins isolate (WPI), β-lactoglobulin, bovine serum albumin, or tryptic soy broth (TSB) and were incubated in bacterial suspensions in modified Welshimer's broth. After 4 h, the number of adherent cells was dependent on the coating, as follows: TSB (10(7) CFU/ml) > bare PS > β-lactoglobulin > bovine serum albumin ∼ WPI (10(4) CFU/ml). The sessile cell counts increased up to 24 h, reaching > 10(7) CFU per peg for all surfaces (P > 0.1), except for WPI-coated PS; this indicates that the inhibitory effects of milk protein conditioning films are transient, slowing down the adhesion process. The 4-h bacterial adhesion on milk protein-coated PS in modified Welshimer's broth supplemented with salt (0 to 10% [wt/vol]) did not vary (P > 0.1), indicating that conditioning with milk proteins was the major determinant for inhibition of bacterial adhesion and that the synergetic effect of salt and milk proteins on adhesion was minimal. Moreover, the presence of 5 to 10% salt significantly inhibited 24-h biofilm formation on the TSB-coated and bare PS, with a decrease of >3 log at 10% (wt/vol) NaCl and almost completely depleted viable sessile bacteria on the milk protein-coated PS.