Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens

The present study evaluates the antimicrobial susceptibility of persister cells of Bacillus cereus and Pseudomonas fluorescens after their regrowth in suspension and as biofilms. Two conventional (benzalkonium chloride—BAC and peracetic acid—PAA) and two emerging biocides (glycolic acid—GA and glyoxal—GO) were selected for this study. Persister cells resulted from biofilms subjected to a critical treatment using the selected biocides. All biocide treatments developed B. cereus persister cells, except PAA that effectively reduced the levels of vegetative cells and endospores. P. fluorescens persister cells comprise viable and viable but non-culturable cells. Afterwards, persister cells were regrown in suspension and in biofilms and were subjected to a second biocide treatment. In general, planktonic cultures of regrown persister cells in suspension lost their antimicrobial tolerance, for both bacteria. Regrown biofilms of persister cells had antimicrobial susceptibility close to those regrown biofilms of biocide-untreated cells, except for regrown biofilms of persister P. fluorescens after BAC treatment, which demonstrated increased antimicrobial tolerance. The most active biocide against persister cells was PAA, which did not promote changes in susceptibility after their regrowth. In conclusion, persister cells are ubiquitous within biofilms and survive after critical biocide treatment. The descendant planktonic and biofilms populations showed similar properties as the original ones.

Extended Downhole Protection by Preservative Biocides as Demonstrated in High Pressure, High Temperature Bioreactors

Preservative biocides are designed to control microbial growth and biogenic souring in the downhole environment. We report the prevention of biogenic souring by 4,4-dimethyloxazolidine (DMO, a preservative biocide) and glutaraldehyde as compared to that afforded by tributyl tetradecyl phosphonium chloride (TTPC, a cationic surface-active biocide), in a first-of-its kind suite of High Pressure, High Temperature (HPHT) Bioreactors that simulate hydraulically fractured shale reservoirs. The design of these new bioreactors, which recreate the downhole environment (temperatures, pressures, formation solids, and frac additives) in a controlled laboratory environment, enables the evaluation of biocides under field-relevant conditions. The bioreactors receiving either no biocide treatment or treatment with a high concentration of TTPC (50 ppm active ingredient) rapidly soured within the first two weeks of shut-in, and all surpassed the maximum detectable level of H2S (343 ppm) after the addition of live microbes to the reactors. Conversely, a higher loading of DMO (150 pppm active ingredient) maintained H2S concentrations below the minimum dectable level (5 ppm) for six weeks, and held H2S concentrations to 10.3 +/- 5.2 ppm after fifteen weeks of shut-in and two post shut-in microbial rechallenges. In a second study, a lower concentration of DMO (50 ppm active ingredient) maintained H2S concentrations below the minimum detectable level through the addition of live microbes after three weeks, and H2S concentrations only registered above 10 ppm upon a second addition of live microbes after five weeks. In this same study (which was performed at moderate temperatures), a 50 ppm (active ingredient) treatment of glutaraldehyde also maintained H2S concentrations below the minimum detectable level through the addition of live microbes after three weeks, and H2S concentrations registered 15.0 +/- 9.7 ppm H2S after four weeks. Similar time scales of protection are observed for each treatment condition through the enumeration of microbes present in each reactor. The differentiation in antimicrobial activity (and specifically, prevention of biogenic souring) afforded by DMO and glutaraldehyde suggests that such nonionic, preservative biocides are a superior choice for maintaining control over problematic microorganisms as compared to surface-active biocides like TTPC at the concentrations tested. The significant duration of efficacy provided by DMO and glutaraldehyde in this first-of-its-kind suite of simulated reservoirs demonstrates that comprehensive preservation and prevention of biogenic souring from completion through to production is feasible. Such comprehensive, prolonged protection is especially relevant for extended shut-ins or drilled but uncompleted wells (DUCS) such as those experienced during the COVID-19 pandemic. The environment simulated within the bioreactors demonstrates that the compatibility afforded by a preservative biocide offers downhole protection that cationic, surface-active biocides do not.

Long-term biocide efficacy and its effect on a souring microbial community

Reservoir souring, which is the production of H 2 S mainly by sulfate-reducing microorganisms (SRM) in oil reservoirs, has been a long-standing issue for the oil industry. While biocides have been frequently applied to control biogenic souring, the effects of biocide treatment are usually temporary, and biocides eventually fail. The reasons behind biocide failure and the long-term response of the microbial community remain poorly understood. In this study, one time biocide treatments with glutaraldehyde (GA) and an aldehyde-releasing biocide (ARB) at low (100 ppm) and high (750 ppm) dosages were individually applied to a complex sulfate-reducing microbial community, followed by one-year monitoring of the chemical responses and the microbial community succession. The chemical results showed that souring control failed after 7 days at 100 ppm dosage regardless the biocide type, and that lasting souring control for the entire one-year timespan was only achieved with ARB at 750 ppm. Microbial community analyses suggested that the high dosage biocide treatments resulted in one order of magnitude lower average total microbial abundance and average SRM abundance compared to the low dosage treatments. The recurrence of souring was associated with reduction of alpha diversity and with long-term microbial community structure change, thus monitoring changes in microbial community metrics may serve as early warnings of the failure of a biocide-based souring control programme in the field. Furthermore, spore-forming sulfate reducers ( Desulfotomaculum and Desulfurispora ) were enriched and became dominant in both GA-treated groups, which could cause challenges to the design of long-lasting remedial souring control strategies. IMPORTANCE Reservoir souring is a detrimental problem for the oil and gas industry as H 2 S corrodes the steel infrastructure, downgrades the oil quality and poses substantial risks to the field personnel and the environment. Biocides have been widely applied to remedy souring, yet the long-term performance of biocide treatments is hard to predict or optimise due to limited understanding of the microbial ecology affected by biocide treatment. This study investigates the long-term biocide performance and associated changes in the abundance, diversity and structure of the souring microbial community, thus advancing the knowledge towards a deeper understanding of the microbial ecology of biocide-treated systems, and contributing to the improvement of current biocide-based souring control practices. The study showcases the potential application of incorporating microbial community analyses to forecast souring and highlights the long-term consequences of the biocide treatment on the microbial communities, with relevance to both operators and regulators.

Microbial Ecology Metrics to Assess the Effect of Biocide on Souring Control and Improve Souring Modelling

Reservoir souring is a long-standing issue for the oil and gas industry caused by sulfate-reducing microorganisms (SRM) producing H2S from sulfate ions. In this work, we investigated the connections between the development of souring and the change in three key microbial ecology metrics: the abundance, alpha diversity and community structure of a souring microbiota under the biocide treatment of 100 ppm glutaraldehyde (henceforth referred to as GA). These are studied in sand-packed flow-through bioreactors during and after the biocide treatment using cutting-edge DNA assays. Our study suggests that the rebound of microbial sulfide production after the 100 ppm GA treatment is closely associated with the recovery in microbial abundance and microbial alpha diversity. The study also shows that 100 ppm GA treatment may lead to a measurable shift in the SRM community structure. By comparing the effluent microbial community with the sand microbial community, the study suggests that the change in alpha diversity of the produced water microbial community might be an early warning for the sulfide breakthrough due to souring recurrence in practice. This work explores the relationship between souring and the underlining microbial community behaviours in response to the 100 ppm GA treatment and, to characterise these changes, we propose measurable metrics. A conceptual model is also proposed describing the near-term biological process behind the biocide treatment-recovery cycle in a souring scenario. Finally, this work highlights the potential applications and caveats of harnessing the increasingly available field microbial community data for the improvement of souring modelling and field souring control strategies.

Nutrient Level Determines Biofilm Characteristics and Subsequent Impact on Microbial Corrosion and Biocide Effectiveness

ABSTRACT The impact that nutrient level has on biofilm characteristics, biocide effectiveness, and the associated risk of microbiologically influenced corrosion (MIC) was assessed using multispecies biofilms from two different oilfield consortia. A range of microbiological, microscopy, and corrosion methods demonstrated that the continuous flow of nutrients for the microbial growth resulted in higher activity, thickness, and robustness of the biofilms formed on carbon steel, which induced greater localized corrosion compared to biofilms formed under batch, nutrient-depleted conditions. Despite of the differences in biofilm characteristics, biofilms displayed comparable susceptibilities to glutaraldehyde biocide, with similar log10 reductions and percent reductions of microorganisms under both nutrient conditions. Nevertheless, nutrient replenishment impacted the effectiveness of the biocide in controlling microbial populations; a higher concentration of cells survived the biocide treatment in biofilms formed under a continuous flow of nutrients. Complementary DNA-/RNA-based amplicon sequencing and bioinformatics analysis were used to discriminate the active within the total populations in biofilms established at the different nutrient conditions and allowed the identification of the microbial species that remained active despite nutrient depletion and biocide treatment. Detection of persistent active microorganisms after exposure to glutaraldehyde, regardless of biofilm structure, suggested the presence of microorganisms less susceptible to this biocide and highlighted the importance of monitoring active microbial species for the early detection of biocide resistance in oil production facilities. IMPORTANCE Microbiologically influenced corrosion (MIC) is a complex process that generates economic losses to the industry every year. Corrosion must be managed to prevent a loss of containment of produced fluids to the external environment. MIC management includes the identification of assets with higher MIC risk, which could be influenced by nutrient levels in the system. Assessing biofilms under different nutrient conditions is essential for understanding the impact of flow regime on microbial communities and the subsequent impact on microbial corrosion and on the effectiveness of biocide treatment. This investigation simulates closely oil production systems, which contain piping sections exposed to continuous flow and sections that remain stagnant for long periods. Therefore, the results reported here are useful for MIC management and prevention. Moreover, the complementary methodological approach applied in this investigation highlighted the importance of implementing RNA-based methods for better identification of active microorganisms that survive stress conditions in oil systems.

Bacterial Activity in a Deposit from a Residual Injection Water Pipeline

This research was conducted on a deposit sampled from a residual injection water pipeline, where corrosion problems identified on metallic equipment have been suspected to be caused by microbiological activity. The residual injection water was also analyzed to confirm the bacterial activity inside the pipeline. Microbiological activity is internationally recognized as a significant contributor to corrosion problems of oilfield equipment, being characterized by rapidity, severity and localized nature. The commonly encountered deposits in water injection pipelines include salts, corrosion products, microbiological mass, suspended matter and water. MIC mitigation methods, as biocide treatment, cannot be implemented without evaluation and confirmation of bacterial activity in the deposit. Several techniques were performed, as SEM and EDS analysis of the deposit to characterize the morphology and elemental composition and microbiological analysis and microscopic examination of the biofilm contained by the deposit, which demonstrated the presence of bacteria communities responsible for MIC.

Biocide Treatment of Invasive Signal Crayfish: Successes, Failures and Lessons Learned

Signal crayfish, as an invasive alien species in Europe, have caused impacts on aquatic communities and losses of native crayfish. Eradication of recently established populations may be possible in small ponds (<2.5 ha) and short lengths of small watercourses using a nonselective biocide. Between 2004 and 2012, a total of 13 sites in the U.K. were assessed for suitability. Six were treated with natural pyrethrum and crayfish were successfully eradicated from three. In Norway, five sites were assessed and two sites were treated with a synthetic pyrethroid, cypermethrin, both successfully. In Sweden, three sites were treated with another synthetic pyrethroid, deltamethrin, all successfully. Defining the likely extent of population was critical in determining the feasibility of treatment, as well as the ability to treat the whole population effectively. Important constraints on projects included site size, habitat complexity, environmental risks, cooperation of landowners and funding availability. Successful projects were manageably small, had good project leadership, had cooperation from stakeholders, had access to resources and were carried out within one to three years. Factors influencing success included treating beyond the likely maximum geographical extent of the population and taking care to dose the treated area thoroughly (open water, plus the banks, margins, inflows and outflows). Recommendations are given on assessing the feasibility of biocide treatments and project-planning.

Behavior of Foodborne PathogensListeria monocytogenesandStaphylococcus aureusin Mixed-Species Biofilms Exposed to Biocides

ABSTRACTIn nature and man-made environments, microorganisms reside in mixed-species biofilms, in which the growth and metabolism of an organism are different from these behaviors in single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms, leading to health risk for humans. Here, we developed two mixed five-species biofilms that included one or the other of the foodborne pathogensListeria monocytogenesandStaphylococcus aureus. The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼105CFU/cm2. The mixed five-species biofilms as well as the pathogens in monospecies biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments.S. aureusdecreased by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the five-species biofilm and decreased by only 1.1 log cycles. Sessile cells ofL. monocytogeneswere affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated microbial community,S. aureuswas eradicated, while there was no significant effect of the biocide onL. monocytogenesor the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection and to identify the species playing the protective role in microbial communities of biofilms.IMPORTANCEThis study demonstrates that foodborne pathogens can be established in mixed-species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, hereS. aureusandL. monocytogenes, but likely caused by specific members or associations in the mixed-species biofilm. Biocide treatment and resistance are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.