Inhibition and Mechanisms of Isothiazolinone and Layered Double Hydroxide–Sodium Pyrithione with Modified Hydrophobic Resin Membranes Against Pipeline Moss Fouling

Abstract

To address pipeline blockages and corrosion caused by moss, this study evaluates the effectiveness of two treatments, Isothiazolinone (IS) and layered double hydroxide–sodium pyrithione (LDH-SPT) modified hydrophobic resin membranes, in preventing moss growth. Furthermore, we closely examined how IS works at a molecular level to stop moss growth. The sequencing results revealed that the predominant algae identified in the pipeline moss community was a norank species of Trebouxiophyceae, accounting for 75.79%. Tests show that IS has strong moss inhibition. It works at low doses (0.2%) and becomes even more effective as the concentration increases. Furthermore, IS remains highly effective at inhibiting moss within a modified hydrophobic resin membrane, but its corrosion resistance is poor. The LDH-SPT@IS composite modified hydrophobic resin membrane addresses the corrosion problem of using IS alone and still works very well at inhibiting moss. Finally, the mechanism of IS’s inhibition of moss was elucidated based on experiments and existing literature. It functions by disrupting moss cellular DNA structure and interfering with the mitochondrial electron transport chain. This research provides the basis for developing efficient, durable, and eco-friendly solutions to prevent pipeline corrosion and moss growth, paving the way for new technologies and materials.

1. Introduction

Pipeline corrosion is a widespread issue, resulting in serious accidents such as casing perforation, failure of drilling and production equipment, and pipeline fractures [1]. Coastal installations are long exposed to the dual corrosive environment of marine and industrial atmospheres, facing not only intense ultraviolet radiation but also high salt and high humidity. The air is rich in water vapor, oxygen, and acidic pollutants, and the severe corrosion resulting from this greatly threatens the mechanical properties of structural materials [2,3,4,5]. However, there are still some problems and challenges in the technology for anti-corrosion of water supply and drainage pipelines, both domestically and internationally. Therefore, researching and developing efficient, economical, and environmentally friendly pipeline corrosion prevention technologies has significant practical importance and broad application prospects [6].

Moss, characterized as a type of lower plant community widely distributed in humid environments [7], tends to proliferate, adhere, and spread within various water conveyance, drainage, and industrial circulation pipeline systems. In particular, in the humid regions of southern China, due to the climate characterized by high temperatures, abundant rainfall, and high air humidity throughout the year, moss tends to grow easily on the surface of pipelines. Moss not only damages the pipeline structure through physical erosion, but also engages in complex chemical interactions with the pipeline material, accelerating aging and significantly reducing their actual service life [8,9]. During their metabolic processes, mosses continuously release acidic substances such as organic acids, which corrode metal components and induce rust formation, exacerbating the wear and tear of facilities.

In order to effectively address pipeline corrosion challenges in humid environments and prevent moss attachment, and to avoid problems such as reduced transportation efficiency and a sharp increase in maintenance costs caused by pipeline damage and moss blockage, it is urgent to develop an efficient and lasting inhibition technology. Among the current solutions, the use of inhibitors and functional membranes is the focus of current research. Particularly, IS represents a class of highly effective broad-spectrum heterocyclic biocides. Its molecules exhibit strong electrophilic characteristics, enabling specific interactions with sulfhydryl groups within microbial cells. By oxidizing key functional groups in cytoplasmic and membrane proteins, it rapidly suppresses metabolic activity and inhibits cellular growth and reproduction [10,11]. Furthermore, Huang et al. [12] systematically investigated the individual and combined effects of sodium dichloroisocyanurate (NaDCC) and IS on cyanobacteria, Vibrio nattani, and associated microbial communities. The two compounds demonstrated synergistic effects in suppressing cyanobacterial photosynthesis and disrupting microbial cell membrane integrity, providing crucial insights for IS applications in pipeline moss prevention and control.

The LDH-SPT material, formed by loading sodium pyrithione (SPT) onto layered double hydroxides (LDHs), has been extensively applied in corrosion protection and biofouling prevention in recent years [13,14,15,16,17]. LDH possesses a unique layered structure and anion exchange capacity, enabling it to serve as a carrier for stable SPT loading and controlled release. SPT, as a compound with both antibacterial and corrosion inhibition functions, can effectively inhibit microbial activity. Zhang et al. [18] prepared Mg-Al LDH thin films, and the results showed that the films exhibit excellent corrosion protection performance. In addition, research in related fields has also provided theoretical support for the performance optimization of LDH-SPT materials. Cao et al. [19] studied Zn-Al LDHs with different intercalated anions and found that a variety of interlayer anions exhibit a synergistic effect in terms of corrosion protection; Zhang et al. [20] prepared aspartic acid-modified self-healing Li-Al LDHs on 6N01 aluminum alloy, and the membrane possesses long-term anti-corrosion ability. These studies provide valuable references for the structural design and performance enhancement of LDH-SPT materials.

Therefore, this study takes moss from a certain pipeline in Hainan Province as the research object. Its purpose is to systematically investigate the inhibitory effects of IS, LDH-SPT composite materials, and the modified hydrophobic resin membranes prepared from them on pipeline corrosion and moss adhesion, and to conduct an in-depth analysis of the mechanism of action based on experiments and existing literature, so as to provide a theoretical basis and practical references for the optimization of pipeline moss prevention and corrosion protection technologies in hot and humid areas.

Figure 1 illustrates the application process of IS in metal protection. Initially, tinplate, paint, and IS are used as raw materials, which are combined to prepare an IS membrane. The mechanism of action is that IS can not only block electron transport but also form a hydrogen-bonded network with microbial DNA, thereby inhibiting the growth of microorganisms such as moss. Ultimately, this material can be applied in scenarios including metal protection and pipeline protection.

2. Materials and Methods

2.1. Chemicals and Materials

The moss sample was collected from a pipeline in Hainan Province. BG-11 culture medium were purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd. (Qingdao, China) Synthetic hydrotalcite (Mg₆Al₂(CO₃)(OH)₁₆·4H₂O), sodium 2-mercaptopyridine-N-oxide (C₅H₆NNaOS), and IS were procured from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). SPU9560 hydrophobic resin was supplied by CNOOC Changzhou Paint (Changzhou, China) and Membranes Industry Research Institute Co., Ltd. (Nanjing, China).

2.2. Instruments and Equipment

The morphology of LDH-SPT was characterized using scanning electron microscopy (SEM, Thermo Scientific Apreo 2S, Waltham, MA, USA). Elemental analysis of LDH-SPT was performed with an energy dispersive spectrometer (EDS, GENESIS, Offenbach, Germany). Fourier transform infrared spectroscopy (FT-IR, PerkinElmer Spectrum TWO, Waltham, MA, USA) was employed for structural characterization. The moss adhesion and corrosion conditions on the membrane surface were examined using a metallographic microscope (HJ1, Shenyang, China).

2.3. Preparation of SPT-Intercalated LDH Nanomaterials

A certain amount of synthetic hydrotalcite (Mg₆Al₂(CO₃)(OH)₁₆·4H₂O) was weighed into a crucible and calcined in a muffle furnace at 350 °C for 5 h. Then, 600 mL of CO₂-free deionized water and 100 mL of deprotonated SPT solution were weighed into a three-necked flask. Under N₂ protection, the mixture was stirred at 60 °C, and 10 g of calcined LDH was weighed and added to it. After ultrasonic treatment for 1 h, the reaction was continued for 24 h. After the reaction, the product was centrifuged, washed with deionized water and absolute ethanol, respectively, and then dried to obtain LDH-SPT.

2.4. Preparation of Hydrophobic Algae-Inhibiting Membranes and Samples

In total, 10 g of hydrophobic resin was added to a mixing tank. Subsequently, 2 g of curing agent, 0.24 g of defoamer, and 1.2 g of thinner were added in sequence, and the mixture was stirred until uniform. Hydrophobic anti-algal membranes with concentrations of 0.2%, 0.5%, and 1% were prepared using the following algal inhibitors: LDH-SPT, IS, and LDH-SPT@IS, respectively.

2.5. Method for Identification of Moss

Metagenomic microbial taxonomic sequencing was employed to analyze 18S rRNA or specific functional gene sequences. Utilizing high-throughput sequencing platforms, the variable regions of these genes were determined to investigate microbial diversity, community composition, and abundance variations.

High-throughput sequencing on the Illumina platform requires the ligation of sequencing adapters before the sequencing process can proceed. To achieve the dual objectives of providing identical sequencing primers for different DNA molecules and enabling sample demultiplexing for distinct users, the adapters are designed with index and barcode sequences. Through the combinatorial use of different indices and barcodes, high-throughput sequencing of numerous samples can be performed simultaneously.

To meet the requirements of high-throughput sequencing library construction, a two-round PCR method was optimized and designed, with the library construction process integrated into the PCR process. This enables efficient and rapid completion of sample detection.

(1)

DNA Extraction: DNA was extracted from 2 samples using the E.Z.N.A™ Mag-Bind Soil DNA Kit [21].

(2)

PCR Amplification: During the second round of PCR amplification, Illumina bridge PCR-compatible primers were introduced.

(3)

Library Quality Control and Pooling: Library fragment size was verified by 2% agarose gel electrophoresis, while library concentration was quantified using a Qubit 3.0 Fluorometer. All samples were mixed in equal amounts at a 1:1 ratio.

2.6. Method for Culturing Moss

In the experiment, the standard BG-11 medium was used as the basic nutrient substrate for algal cultivation. The specific preparation method is as follows: Accurately weigh 1.70 g of BG-11 medium powder, dissolve it in 1 L of deionized water, and stir thoroughly until completely dissolved. Place it in an autoclave and sterilize at 121 °C for 15 min to ensure the medium is sterile. After sterilization, the medium is cooled to room temperature for later use. The inoculation procedure was carried out in a laminar flow hood under sterile conditions. Using a pipette, the algal inoculum was transferred into the sterilized BG-11 medium at an inoculation volume ratio of 5–10% [22,23].

The cultivation process was conducted in an artificial climate incubator with strictly controlled environmental parameters: LED lighting maintained at 2000 lux with a 12 h:12 h light–dark cycle; constant temperature at 25 °C; and intermittent manual shaking performed three times daily for 30 s each time to enhance gas exchange and ensure adequate carbon dioxide supply.

During the cultivation period, the growth status of algae was observed regularly at a fixed time every day. The medium was replaced every two weeks. Meanwhile, regular observations were conducted to monitor the growth status of algae and potential contamination.

2.7. Method for Evaluating Inhibitor Performance

We evaluated the moss inhibition efficacy of two inhibitors, IS and LDH-SPT. The initial concentration of the algal solution was set to 3–5 green algal aggregates with a diameter of approximately 1–2 mm per cubic centimeter. IS and LDH-SPT were tested at three concentrations: 0.2%, 0.5%, and 1%. Three experimental groups were established by combining 1% LDH-SPT with each concentration of IS. Each experimental group was set with two parallel samples, and a blank moss culture solution was established as the control.

The experimental samples were placed in an intelligent climate chamber with the following conditions: temperature at 25 °C, light intensity at 2000 lux (light–dark cycle of 12 h:12 h). The samples were cultured continuously for 10 days, and the experimental phenomena were recorded by taking photos every day to observe the survival status of the moss.

2.8. Method for Evaluating Membrane Inhibition Performance

Membrane samples containing IS and LDH-SPT inhibitors were prepared at concentrations of 0.2%, 0.5%, and 1%, respectively. Three experimental groups were established by combining 1% LDH-SPT with each of the three IS concentrations to prepare composite membranes. The anticorrosion and moss inhibition performance of these membranes was evaluated. Each experimental group was set with three parallel samples, and SPU9560 hydrophobic resin samples were used as controls to eliminate the influence of the substrate itself on moss adhesion.

The coated samples were immersed separately in Petri dishes containing moss culture solution, and the Petri dishes were placed in an intelligent artificial climate incubator. The set cultivation conditions were identical to those in the inhibitor evaluation experiment. Experimental phenomena were recorded by taking photos every day to observe the survival status of moss and assess the adhesion amount of moss on the membrane samples.

3. Results

3.1. Characterization of LDH-SPT

The morphology of LDH-SPT is shown in Figure 2a, which is mainly composed of irregular flakes with rough surfaces, and some of them overlap with each other. Figure 2b shows that the sample has a relatively high oxygen content, with a mass fraction of 46.060%. The mass fractions of other elements are as follows: C at 31.404%, N at 15.279%, Mg at 4.639%, Al at 2.464%, and S at 0.154%. The infrared spectrum of LDH-SPT in Figure 3a shows that the wave number at 3451.2 cm⁻¹ is attributed to O-H stretching vibration; 1368.5 cm⁻¹ to the asymmetric stretching vibration of NO₃⁻; 768.2 cm⁻¹ to M-O-H bending vibration (M=Mg, Al) or organic residual vibration; 660.3 cm⁻¹ to M-O-M lattice vibration (M=Mg, Al); and 446.9 cm⁻¹ to M-O bending vibration or lattice vibration. The infrared spectrum of IS in Figure 3b shows that the wave number at 3397.35 cm⁻¹ is attributed to N-H stretching vibration; 1626.4 cm⁻¹ to the stretching vibration of C=O (carbonyl group); 1348.3 cm⁻¹ to the skeletal vibration of aromatic rings or the stretching vibration of C−N bonds; and 708.6 cm⁻¹ to the stretching vibration of C-S-C (thiazole ring).

3.2. Results of Moss Identification

Based on 18S rRNA sequencing analysis, the species abundance of the moss samples is shown in

Table 1. Species abundance of 18S rRNA samples.

Species	Percentage (%)
norank Trebouxiophyceae	75.79
Adineta	0.73
Jaagichlorella	14.76
Jenufa	0.04
Tripylina	0.01
Klebsormidium	0.01
Amblydorylaimus	0.01
Orbilia	7.0 × 10−3
Bradymyces	2.54
Pichia	1.34
Rigidohymena	3.5 × 10−3
Sterkiella	1.8 × 10−3

. The most dominant algal group was norank Trebouxiophyceae (75.79%) within the Chlorophyta phylum. Jaagichlorella represented the second most abundant genus at 14.76%, which also belongs to the Chlorophyta phylum. These results indicate that the sample constitutes a community overwhelmingly dominated by green algae.

3.3. Investigation of Inhibitor Performance

In the inhibitor screening experiment, the comparison shown in Figure 4(b₁–b₃) demonstrates that IS exhibits significant inhibition effects on common moss species even at low concentrations. Obvious chlorophyll degradation was observed after 1 day of treatment, which was manifested as the color of the moss changing from green to brown. The inhibitory effect improved with the increase in concentration. In Figure 4(c₁–c₃), the three concentrations of LDH-SPT showed no obvious inhibitory effect on the moss. In Figure 4(d₁–d₃), the combination of LDH-SPT and IS exhibited an inhibitory effect on the moss, but its action rate was slower than that of IS alone. For the composite inhibitors of LDH-SPT (1%)@IS (0.5%, 1%), the color change of the moss from green to brown was observed after 4 days of the experiment, while for the composite inhibitor of LDH-SPT (1%)@IS (0.2%), this color change was only observed after 8 days of the experiment.

IS can rapidly disrupt cellular metabolism, leading to rapid degradation of chlorophyll, thus taking effect quickly even at low concentrations. LDH-SPT itself has weak moss-inhibiting activity, so its effect is not obvious when used alone. When compounded with IS, the carrier structure of LDH-SPT may slow down the transfer rate of IS to cells through adsorption or sustained-release effects, thereby reducing its action speed. This results in the characteristic that the inhibitory effect still exists but takes effect more slowly.

3.4. Investigation of Membrane Inhibition Performance

Adhesion testing was conducted in accordance with the GB/T 9286-2021 standard [24]. Using a 1 mm grid spacing and tape peel-off method, no peeling of the paint membrane was observed, with smooth and regular cutting edges and no issues such as flaking or chipping. For impact resistance testing, a 1 kg hammer with a 10 mm diameter was dropped from a height of 50 cm onto the composite membrane. After the test, the paint membrane remained intact, with no cracks, damage, or detachment. This demonstrates good mechanical properties of the composite membrane.

In the membrane inhibition experiment, Figure 5(b₁–b₃) demonstrates that compared to the blank membrane, all three IS-containing membranes effectively prevented moss attachment. However, they exhibited poor corrosion resistance with noticeable membrane detachment. Figure 5(c₁–c₃) reveals that the LDH-SPT membranes at all three concentrations showed moss attachment levels similar to the control group, but displayed excellent corrosion protection without any membrane deterioration. Figure 5(d₁–d₃) indicates that the composite membranes containing 1% LDH-SPT combined with three different IS concentrations showed no signs of membrane detachment. Side rust is corrosion caused by incomplete coverage of the membrane. Specifically, the LDH-SPT (1%)@IS (0.2%, 0.5%, 1%) composite membranes exhibited virtually no moss attachment. Further observation via the metallographic microscope in Figure 6 reveals that no moss adhered to the IS membrane, while a trace amount of moss adhered to the LDH-SPT (1%)@IS (1%) composite membrane, which was far less than that on the LDH-SPT membrane. When observing membranes using the upright light source of a metallographic microscope, it was found that purple and green colors are usually caused by light interference effects (especially thin-film interference) if the membrane is thin and has a smooth surface. The unevenness of membrane thickness or changes in microstructure lead to different light interference conditions in different regions, thus presenting different colors.

Figure 7(a₁,a₂) show the SEM images of the cross-section of the membrane before and after the experiment, respectively. The cross-section of the membrane maintained an overall continuous structure, with no large-scale cracking or detachment observed; only slight interfacial changes were noted. Combining Figure 7(b₁,b₂), the EDS mapping results show that the distribution and content of key elements such as Al, Mg, N, and S did not undergo significant changes before and after the evaluation. This phenomenon indicates that during the evaluation process, the chemical composition and elemental distribution of the composite membrane did not exhibit noticeable degradation or loss, nor did the bonding interface between the membrane and the substrate suffer structural damage. These results confirm the excellent stability of the membrane at the microscopic level, providing strong evidence for the long-term anti-corrosion and sustained algae inhibition performance of the membrane.

EIS tests were conducted on three different types of composite membranes after experimentation. Figure 8a exhibits an impedance magnitude of 10⁷ ohm, which is the highest among the three. This indicates that the membrane has the strongest electrolyte barrier property and relatively optimal anti-corrosion performance. However, based on previous experimental findings, when the IS concentration is 0.2%, its algae inhibition effect is comparatively the weakest among the three. Figure 8c shows an impedance magnitude of 10⁵ ohm, the lowest among the three. This suggests that the membrane has the weakest electrolyte barrier property and relatively the poorest anti-corrosion performance. Yet, when the IS concentration is 1%, its algae inhibition effect is the strongest among the three. Figure 8b displays an impedance magnitude of 10⁶ ohm, lying between that of 8a and 8c. Membranes with impedance below 10⁶ ohm are generally considered to have failed. Therefore, with an IS concentration of 0.5%, this formulation represents a relatively balanced ratio between “anti-corrosion” and “algae inhibition” performance.

3.5. Inhibition Mechanism of IS-Based Membranes

The IS molecule has strong electrophilic properties, enabling it to specifically interact with sulfhydryl groups in microbial cells. By oxidizing key functional groups in cytoplasm and membrane proteins, it rapidly inhibits cellular metabolic activity and growth as well as reproduction [12,25].

In terms of molecular action mechanism, IS is a typical electrophilic biocide. Due to the active heterocyclic ring in its molecule, it can specifically bind to intracellular biological macromolecules. The mechanism diagram of IS inhibition in Figure 9 shows that the active groups of IS can form a stable hydrogen bond network with the bases in DNA molecules. This intermolecular force leads to the selective adsorption of IS on the cell surface, which further attacks the nucleophilic centers in the cell and ultimately destroys the secondary structure of DNA [12]. Such structural damage severely disrupts the normal replication and transcription processes of genetic material, resulting in the loss of fundamental physiological functions and metabolic activity in cells.

Furthermore, IS exhibits strong cell membrane penetration capability [26], enabling it to rapidly traverse cell wall and membrane barriers to directly target the electron transport chain on the mitochondrial inner membrane, as illustrated in Figure 9. By inhibiting the activity of key components such as the NADH dehydrogenase complex and cytochrome oxidase, IS disrupts electron transfer processes, consequently interfering with oxidative phosphorylation and ultimately leading to cellular energy metabolism dysfunction [27].

After compounding IS with LDH-SPT, from the perspective of the carrier structure of LDH-SPT, LDH materials possess a typical layered two-dimensional nanostructure [28], with regulable ion exchange channels and adsorption sites between layers. This structure enables it to not only serve as a loading carrier for SPT but also form multiple interactions with IS. On one hand, the metal cations between LDH layers can form coordination bonds with polar groups such as nitrogen and oxygen in IS molecules, anchoring part of IS in the interlayer gaps; on the other hand, the layered stacking of LDH creates a physical barrier to the diffusion path of IS, preventing it from being released into the system as rapidly as when used alone.

4. Conclusions

This study systematically investigated the inhibition performance of IS and LDH-SPT inhibitors and their modified hydrophobic resin membranes against pipeline-adhered moss, along with the moss inhibition mechanism of IS. Through high-throughput sequencing of pipeline moss samples from Hainan Province, the dominant species was identified as a norank species of Trebouxiophyceae, with a relative abundance as high as 75.79%.

In the study on moss-inhibiting performance, IS exhibited significant inhibitory effects, while LDH-SPT showed no obvious moss-inhibiting effect. However, the LDH-SPT@IS composite inhibitor demonstrated a distinct moss-inhibiting effect. In modified hydrophobic resin membrane applications with three concentrations (0.2%, 0.5%, and 1%), the IS membrane displayed excellent anti-adhesion performance with no moss attached to its surface, but its anti-corrosion effect was poor. LDH-SPT membrane showed excellent anti-corrosion performance but had no obvious moss-inhibiting effect. The experiment confirmed that the LDH-SPT@IS composite membrane significantly improved the anti-corrosion effect of the membrane.

Through its strong electrophilic properties, IS disrupts the secondary structure of cellular DNA and inhibits the electron transport chain on the inner mitochondrial membrane, thereby interfering with genetic information transmission and energy metabolism to achieve the inhibition of moss. This study provides theoretical support and material design directions for the development of efficient and environmentally friendly pipeline moss-prevention technologies, and offers guidance for the selection and optimization of moss-prevention materials in practical applications.