There are challenges of microbial contamination in hydraulic fracturing, whereby microbes can lead to souring, corrosion, and biofouling in shale reservoirs. It is important to use an optimized biocide program to control these issues. This article details development and evaluation of high pressure/high temperature (HPHT) bioreactors to test biocide effectiveness under reservoir conditions.
KEN Wunch, LANXESS Material Protection Products
During hydraulic fracturing, upwards of 5 million gal of water from the topside is injected into the shale reservoir, to fracture the rock and release hydrocarbons. For perspective, non-potable frac water can easily have 104 bacteria/ml (10,000/ml), representing thousands of different species.
As a result, approximately 400 hundred trillion bacteria (4 × 1014 – 400,000,000,000,000) are injected into the reservoir per each well fraced. Since pore spaces in shale formations are extremely tight and it’s generally considered that the native, endogenous microbial population in a shale reservoir is extremely limited or non-existent, the dominant bacterial population in these reservoirs is exogenous and introduced during the fracturing process.
The Dutch botanist and microbiologist Lourens Baas Becking developed the hypothesis that "Everything is everywhere, but the environment selects." The environment in a shale reservoir can be very inhospitable for microbial growth, with temperatures often above 100°C, salinities above 10%, and pressures above 5,000 psi. Assuming 99.99% of the injected topside bacteria die in these conditions, 40 billion potentially viable bacteria still could survive in these reservoirs. The key question associated with this statement is, what microbes and metabolic processes is the environment in the shale reservoir selecting for? Unfortunately, some of the answers are souring, corrosion and biofouling, Fig. 1.
The first method of adaption for organisms that have been introduced into the hostile environment of the shale reservoir is the formation of a biofilm. Microbes settle on solid substrates, including metal and geological surfaces, as well as proppants. These microorganisms secrete extracellular polymeric substances (EPS), which form a protective matrix that encapsulates the microbial community, allowing it to thrive in harsh conditions.
Over time, the biofilm can become a thick, slimy layer that can impede fluid and gas flow, harbor sulfidogenic microbes, and promote localized corrosion. Evidence of the damage a biofilm can exert in a hydraulically fracked shale reservoir is detailed in Wunch & Silva (2023). Figure 2 illustrates how biofilms can block pore spaces between proppants with a lab-demonstrated conductivity loss of ~17%. Using industry modeling software, this conductivity loss translates to ~6,000 bbl of oil deferred during the first six months of production.
Microbially Influenced Corrosion (MIC) occurs when biofilms form on metal surfaces, leading to metabolically driven corrosion that can cause equipment and pipeline failures. Reservoir or systemic souring arises when sulfate- or thiosulfate-reducing microbes “breathe in” sulfate or thiosulfate and “breathe out” hydrogen sulfide (H₂S), a highly toxic and corrosive molecule. In oil and gas operations, H₂S is particularly problematic, as it threatens worker health and safety, contaminates extracted hydrocarbons, and reacts with iron in reservoirs or equipment to form iron sulfide (FeS).
FeS is an oleophilic scale that can promote under-deposit corrosion, complicate oil/water separation, and combine with organic deposits to foul and plug equipment and wells. The biological production of sulfide and the resulting MIC not only disrupts oil and gas operations but also has severe environmental consequences. For example, Loss of Primary Containment (LOPC), due to MIC, has led to several hydrocarbon spills, with the 2006 Alaskan oil spill being particularly notorious, as it released 267,000 gal (about 6,357 bbl) of oil into the sensitive Arctic tundra. Furthermore, removing sulfides from refined hydrocarbons is energy-intensive and has a significant carbon footprint. Inefficient removal can lead to the release of sulfur dioxide during combustion, exacerbating environmental impacts.
These deleterious microbiological activities cost the oil and gas industry billions of dollars annually and can threaten health, safety and the environment. As a result, risk mitigation is extremely important to the hydraulic fracturing industry and the first line of a proactive defense is the introduction of biocides during completion activities. Biocides are used in hydraulic fracturing operations to control the growth of contaminant microorganisms that lead to corrosion, souring, and conductivity loss. A variety of biocides are utilized and can be classified by mechanism of action, speed of kill, and the length of residual activity.
BIOCIDES UTILIZED IN HYDRAULIC FRACTURING
Biocides are generally categorized into two broad classes: oxidizers and non-oxidizers. The mechanisms of action, application strategies, monitoring methods, and responses to system contamination differ significantly between these two groups. Oxidizing biocides, such as sodium hypochlorite, chlorine dioxide, and peracetic acid, exhibit broad-spectrum activity against a wide range of microorganisms. They are generally fast-acting (within minutes), highly reactive, and possess multiple modes of action for microbial deactivation (Nalepa & Williams, 2010). However, this high reactivity makes them prone to rapid depletion during operations and can lead to corrosion of infrastructure, if residual levels are not carefully controlled.
In contrast, non-oxidizing biocides are more targeted in their action, primarily disrupting the microbial cell membrane or inhibiting key cellular processes, rather than causing oxidative damage. These biocides are typically small, organic molecules that are less reactive than oxidizers and are compatible with most completion or production chemistries. Non-oxidizers tend to act more slowly (taking hours to days), exhibit longer persistence in the reservoir, and have lower corrosivity. Due to these characteristics, non-oxidizing chemistries are the most commonly applied during the completion operations of hydraulic fracturing.
Non-oxidizing biocides are classified as either surface-active or electrophilic. Surface-active biocides used in oil and gas operations are often cationic quaternary ammonium and phosphonium compounds (Quats), such as alkyl dimethyl benzyl ammonium chloride (ADBAC), didecyl dimethyl ammonium chloride (DiDAC) and tributyl tetradecyl phosphonium chloride (TTPC). These biocides primarily function by disrupting the microbial cell membrane, compromising cell integrity, and leading to microbial death. Originally developed as hard surface disinfectants, they are particularly effective at mitigating established biofilms. However, the ionic nature of these molecules can cause compatibility and deactivation issues with charged molecules, such as partially hydrolyzed polyacrylamide (hPAM), which is commonly used as a friction reducer in slick-water operations. Additionally, these actives can also be adsorbed onto rock or proppant, leading to further biocide deactivation (Moore et al., 2017).
Electrophilic biocides function by forming covalent bonds with nucleophilic sites on microbial cells, such as proteins or DNA, inhibiting critical cellular processes and causing microbial inactivation. These biocides are commonly employed in hydraulic fracturing, where their selection is based on treatment cost and the required duration of protection. Given that hydraulic fracturing operations typically involve minimal-to-no water replenishment after completions and drill-out, the focus of biocide selection is on a one-time application that provides cost-effective, long-lasting protection.
Commonly used electrophilic biocides in hydraulic fracturing include:
DBNPA (2,2-dibromo-3-nitrilopropionamide)
Glut (glutaraldehyde)
THPS (tetrakis [hydroxymethyl] phosphonium sulfate)
Bronopol (2-bromo-2-nitro-1,3-propanediol)
THNM (tris[hydroxymethyl]nitromethane)
DMO (4,4-dimethyloxazolidine)
In general, rapid-acting biocides, such as DBNPA, along with the aforementioned oxidizers and surface active biocides, inactivate bacteria quickly but provide little to no residual activity. Glut, THPS and bronopol react more slowly and offer some residual activity. However, THPS is cationic and can reduce the efficiency of commonly used friction reducers, making it less favored during completions. Preservatives, such as DMO and THNM react very slowly but provide residual activity that can last for weeks or months within the reservoir.
Ultimately, a completion engineer must carefully consider two critical questions when developing a microbial control strategy: What is the most valuable component of my system? And what is the most effective microbial control program to safeguard it? If the primary concern is protecting completions equipment and topside tanks, then quick-kill chemistries may be viable solutions. However, if the goal is to protect the hydrocarbon reservoir itself, where microbial contamination can cause issues, such as souring or reduced production efficiency, a more proactive long-term strategy is necessary. In this case, chemistries designed for sustained microbial control in the reservoir can be evaluated through LANXESS’ bioreactor program.
DEVELOPMENT OF HPHT BIOREACTORS
The selection of hydraulic fracturing biocides is usually driven by experimental results generated under benchtop conditions of ambient temperature and pressure. Adenosine triphosphate (ATP) testing has supplanted traditional culture-based methods (e.g. “bug bottles”), due to ease of use and rapid turnaround; however many laboratories depend solely on studies that demonstrate the ability of a biocide to rapidly (minutes-hours) reduce the level of ATP in a water sample. This over-reliance on a rapid method underestimates the capabilities of slower-acting preservative biocides. In addition, testing of water samples, only, may not give a complete understanding of the behavior of a biocide under conditions encountered in the reservoir, such as contact with proppant, shale, or hydraulic fracturing additives, such as friction reducer.
LANXESS’ research laboratory in Wilmington, Del., has developed a pioneering suite of HPHT bioreactors (Fig. 3) specifically designed to evaluate the performance of biocides under the extreme downhole conditions encountered during hydraulic fracturing (Ferrar et al., 2021a). Each bioreactor has a 500-mL internal volume and is filled with shale formation solids, proppants, and key completion chemistries, such as friction reducers, biocides, and scale inhibitors. The reactors are inoculated with field-relevant sulfate- and thiosulfate-reducing microbial populations, capable of thriving and producing H₂S under the variable temperature and pressure conditions typical of downhole environments.
Importantly, the temperature limitations in these studies are not due to engineering constraints of the bioreactors, but rather the physiological challenges of maintaining a stable microbial population above 60°C in the laboratory. The systems can replicate reservoir conditions, achieving temperatures up to 150°C and pressures up to 3,000 psi. Constructed from titanium and SS316L, these closed systems enable direct measurement of H₂S concentrations in simulated fracturing fluid, as well as the determination of viable microbial cell counts.
Water used during completions is not the only source of microbial contamination in the reservoir. The coiled tubing used for drilling out plugs can also introduce fluids that contaminate the reservoir. Additionally, densely drilled wells can interact with one another, leading to the migration of fracturing fluids from one well to another. This can result in damage, loss of pressure, or contamination of a nearby well's reservoir with microbes (frac hit). To simulate microbial contamination events that occur after the initial drilling and fracturing, microbial rechallenges are routinely conducted in the bioreactors on days 21 and 35, without the addition of further mitigation chemistries. This approach enables a more accurate assessment of the longevity of microbial control technologies in the reservoir.
EVALUATION OF BIOCIDES IN THE HPHT BIOREACTOR PROGRAM
Our first comprehensive study using the HPHT hydraulic fracturing bioreactors (Ferrar et al., 2021a) focused on three commonly used electrophilic biocides with different duration profiles and included a fast-acting Quat (TTPC), an active with intermediate duration (Glut), and a preservative (DMO). Triplicate reactors were inoculated with Wolfcamp shale, low-iron sand proppant, and a sulfidogenic field culture. Sterile, simulated fracturing fluid (SSFF) was injected on day 0 containing 3% TDS water, 3.5 g/L lactate, 1,000-ppm anionic hPAM and 50-ppm (active ingredient – a.i.) biocide. The reactors were run in triplicate at 30°C and re-challenged on day 21 (simulating drill out) and day 35 (simulating frac hit). Results of the study are presented in Fig. 4.
This study details the findings, concluding that both Glut and DMO effectively suppressed biogenic H₂S production and the recovery of viable microbes from the bioreactors for at least 4–5 weeks at low dosing levels. Glut began to lose control of sulfide production after the second inoculation of bacteria at week 4, while DMO lost control following the third bacterial re-challenge at week 5. Additionally, the study showed that DMO inhibited both souring and microbial viability for over 4 months at field-relevant biocide dosing levels (150 ppm a.i.). This makes DMO an excellent choice for long-term microbial control in reservoirs to mitigate souring, biofouling, and corrosion.
As hypothesized, the Quat (TTPC) lost control of the microbial population before the first re-challenge, around 10 days. This was likely due to either interactions between TTPC and hPAM, proppant, or shale, or the inherent characteristics of fast-acting biocides that kill quickly but offer no residual control. Regardless, these HPHT bioreactor results suggest that quats are a poor choice for prolonged microbial control in reservoirs.
The next phase in the evolution of our HPHT Bioreactor Program involved developing a field population that could be routinely cultured at reservoir-relevant temperatures (60°C), as detailed in Yin et al. (2025, in-press). In this study, the reactors were initially inoculated with a mesophilic culture (optimal growth at 30°C), simulating ambient topside temperatures, and then re-challenged with a thermophilic culture (optimal growth at 60°C), simulating reservoir conditions. Following the initial inoculation with the mesophilic culture, the HPHT bioreactor temperatures were gradually ramped up from 30°C to 60°C, to mimic the temperature increase of injection fluids during hydraulic fracturing. Both 50 ppm (a.i.) of DMO and DiDAC (another fast-acting quat) were tested. The results, outlined in the paper, again show the failure of the quat, with a loss of population control around Day 4. In contrast, DMO maintained control through the thermophilic culture challenge, only losing control shortly before week 5. These results again highlight DMO as the superior chemical choice for preserving reservoir conditions.
RELEVANCE TO FIELD APPLICATION
Validating laboratory data in real-world field conditions is the ultimate test for any laboratory program. To compare with the data from our HPHT Bioreactor Program, we conducted a multi-year field study in the Vaca Muerta basin, Argentina, focused on assessing the performance of various preservative chemistries. (Silva, et al., 2023). The selected field contained high-density, shale gas wells with approximately 65-80 fracturing stages in the horizontal legs.
Prior to 2019, the microbial control program in the Vaca Muerta basin used a non-oxidizing biocide program. In the second half of 2019, the preservative chemistries DMO and THNM were introduced to the basin, and the results of H2S production before and after their implementation are compared in Fig. 5. Approximately 72% of wellhead samples treated with non-preservative biocides exceeded the key performance indicator (KPI) of 2 ppm sulfide, with some wells reaching over 7 ppm H2S. However, following the introduction of DMO and THNM, only two samples (~5%) exceeded the KPI, and neither measurement was above 3 ppm. Most notably, the operator reduced overall biocide consumption by 7%, which corresponded to an impressive 35% decrease in H2S scavenger spend.
GARBAGE IN LEADS TO GARBAGE OUT
In the early years of hydraulic fracturing, wells were typically completed using relatively clean surface water or even potable water, which had very low bacterial loads. However, due to social, regulatory and operational pressures, most of the industry has shifted to using produced water or other more contaminated water sources for fracing. As a result, trillions of microbes are introduced into shale reservoirs, with billions potentially thriving and causing damage. Critics have argued that some reservoirs may be too inhospitable for microbial growth. However, studies have shown that metabolically active bacteria have been recovered from formations as hot as ~150°C (Fichter et al., 2012). Moreover, since there is little evidence of a problematic indigenous microbial population in shale reservoirs, controlling the microbial content of injection fluids to prevent biofilm formation in the reservoir is crucial.
The primary goal of any microbial control program for hydraulic fracturing should be to mitigate the garbage being injected and to Protect the Integrity of the Reservoir. While completion equipment can be replaced, the reservoir itself cannot. If the reservoir becomes contaminated, it may continuously produce soured and corrosive hydrocarbons at reduced levels, ultimately damaging production equipment and complicating separation and refining processes.
Since all fluids are injected into the reservoir during the relatively brief completion process and are not replenished, there is only one opportunity to effectively implement a microbial control program. Although retroactive biocide squeezes during production may be possible, their effects are only temporary. Consequently, data from our bioreactor program and field studies have led to recommendations focused on preventing contamination, or "garbage," from entering production fluids by applying the right biocide to protect the reservoir. Furthermore, ineffective and off-spec biocides exacerbate the issue of "garbage in" and undermine confidence in microbial control programs.
HPHT bioreactor data correlate well with our field observations in Argentina and suggest that reservoir and production protection can be achieved for:
Months with preservatives (DMO)
Weeks by glutaraldehyde
Hours/days for oxidizers and quats (DiDAC & TTPC)
Specific recommendations, derived from decades of lab and field experience within the LANXESS microbial control group, are outlined in Table 1. These recommendations, based on empirical evidence, conclude that applying DMO during completions will help mitigate the “garbage” introduced into the reservoir and control microbial risks, as well as manage the "garbage out" in production fluids. WO
REFERENCES
Ferrar, J., Maun, P., Wunch, K., Moore, J., Rajan, J., Raymond, J., Solomon, E., & Paschoalino, M. (2021a). High Pressure, High Temperature Bioreactors as a Biocide Selection Tool for Hydraulically Fractured Reservoirs. SPE Hydraulic Fracturing Technology Conference, SPE-204198-MS.
Ferrar, J., Maun, P., Wunch, K., Moore, J., Rajan, J., Raymond, J., Solomon, E., & Paschoalino, M. (2021b). Extended Downhole Protection by Preservative Biocides as Demonstrated in High Pressure, High Temperature Bioreactors. SPE International Conference on Oilfield Chemistry, SPE-204377-MS.
Fichter, J., Moore, R., Summer, E. & Wunch, K. (2012). How Hot is Too Hot for Bacteria? A Technical Study Assessing Bacterial Establishment in Downhole Drilling, Fracturing and Stimulation Operations. NACE Corrosion Conference, C2012-0001310.
Moore, J., Massie-Schuh, E., Doshi, D., Schultz, C., Castillo, C., Patel, B., Moore, M., Rajan, J., & Ajayi, B. (2017). Oilfield Biocide Performance in the Presence of Shale Formation Rock. SPE International Conference on Oilfield Chemistry, SPE-184583-MS.
Moore, J., Massie-Schuh, E., Wunch, K., & Manna, K. (2019). Insights into Effective Microbial Control Through a Comprehensive Microbiological Audit of Hydraulic Fracturing Operations. SPE International Conference on Oilfield Chemistry, SPE-193606-MS.
Silva, V., Wunch, K., Solomon, E., & Maun, P. (2023). Extended Field Study Tracking the Performance of the Preservative Biocides, THNM and DMO, in Unconventional Wells. SPE International Conference on Oilfield Chemistry, SPE-213862-MS.
Williams, T., & Nalepa, C. J. (2010). Biocides: Selection and Application. In Z. Amjad (Ed.), The Science and Technology of Industrial Water Treatment (Chapter 19). CRC Press, Taylor and Francis Group, Boca Raton, FL.
Wunch, K., & Silva, V. (2023). Protecting the Reservoir from Diminishing Productivity Caused by Downhole Biofilm Growth in Shale Plays. SPE International Conference on Oilfield Chemistry, SPE-213852-MS.
Yin, B., Wunch, K., Maun, P., & Rama, A. (2025). A Study of Sulfide Control in Hydraulic Fracturing Using High Pressure High Temperature Bioreactors. SPE International Conference on Oilfield Chemistry. SPE-224286-MS (In Press).
KEN WUNCH, Ph.D., is a distinguished Energy Technology Fellow with LANXESS in the Material Protection Products business. In his current role, he is responsible for business development, technology transfer, and shaping the innovation pipeline and strategy for global energy applications. Dr. Wunch earned his Ph.D. in Environmental Microbiology and has accumulated over 15 years of experience in the industry. His career includes significant positions at Baker Hughes, BP, and Dow, where he honed his expertise in the development and field application of biocides, corrosion inhibitors, and H2S scavengers. As a global Subject Matter Expert (SME) in oilfield microbiology, bioinformatics, reservoir souring, and microbially influenced corrosion, he has made substantial contributions to the field. He is the author of over 50 publications and patents, including notable books, such as Microbial Bioinformatics in the Oil & Gas Industry and Petroleum Microbiology: The Role of Microorganisms in the Transition to Net Zero Energy. In addition to his research and publications, Dr. Wunch is an esteemed instructor for SPE, where he teaches courses on microbial control in oil and gas applications.