# Hydrogen Sulfide Induced Biodeterioration of Mortars and Cement Paste: A Multi-Scale Investigation using laboratory accelerated test

Janette Ayoub, Marielle Guéguen Minerbe, Tony Pons, Guilhem Simon, Mario Marchetti

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Janette Ayoub, Marielle Guéguen Minerbe, Tony Pons, Guilhem Simon, Mario Marchetti. Hydrogen Sulfide Induced Biodeterioration of Mortars and Cement Paste: A Multi-Scale Investigation using laboratory accelerated test. Results in engineering, 2025, pp.108695. 10.1016/j.rineng.2025.108695. hal-05414924

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Hydrogen Sulfide Induced Biodeterioration of Mortars and Cement
Paste: A Multi-Scale Investigation using laboratory accelerated test

Janette Ayoub, Marielle Guéguen Minerbe, Tony Pons,
Guilhem Simon, Mario Marchetti

PII: S2590-1230(25)04738-3

DOI: https://doi.org/10.1016/j.rineng.2025.108695

Reference: RINENG 108695

To appear in: *Results in Engineering*

Received date: 23 October 2025

Revised date: 2 December 2025

Accepted date: 10 December 2025

Please cite this article as: Janette Ayoub, Marielle Guéguen Minerbe, Tony Pons, Guilhem Simon, Mario Marchetti, Hydrogen Sulfide Induced Biodeterioration of Mortars and Cement Paste: A Multi-Scale Investigation using laboratory accelerated test, *Results in Engineering* (2025), doi: https://doi.org/10.1016/j.rineng.2025.108695

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Highlights

* Accelerated test reliably replicates *in situ* degradation in sewer conditions

* Accelerated test replicates 49-month sewer degradation profiles in only 180 days

* µ-Raman coupled with chemometrics reveals zonation and confirms precise mineral changes

* Degradation is more advanced in mortar than in cement paste sections

<figure><img src="image_3.png" /></figure>
# Hydrogen Sulfide Induced Biodeterioration of Mortars and Cement Paste: A Multi-Scale Investigation using laboratory accelerated test

Janette Ayoub¹*, Marielle Guéguen Minerbe¹, Tony Pons¹, Guilhem Simon², Mario Marchetti³

1 Univ. Gustave Eiffel, MAST-CPDM, Marne-la-Vallée F77454, France

2 Sorbonne Université, CNRS, Laboratoire MONARIS, c49, F75252, Paris, France

3 Univ. Gustave Eiffel, MAST-EMGCU, Marne-la-Vallée F77454, France

*Corresponding author (janette.ayoub@univ-eiffel.fr)

## Abstract

Cementitious materials in sewer environments are exposed to highly aggressive biogenic sulfuric acid, yet their long-term resistance remains difficult to evaluate without extended field trials. This study validates an accelerated test protocol that reproduces up to 49 months of *in situ* degradation in only 190 days under controlled microbial and H₂S exposure. Three binders (CEM I, CEM V/A, and CAC) were tested in both mortar and cement paste forms. Despite higher total porosity, cement paste consistently outperformed mortar in durability due to its denser pore network and greater buffering capacity, highlighting the key role of microstructural organization. Degradation was monitored using multi-scale analytical techniques, including macroscopic indicators (pH, mass, dimensional change), SEM-EDS, and µ-Raman mapping combined with PCA and MCR-ALS, revealing clear zonation patterns and sulfate-rich biodeterioration fronts. Using different specimen dimensions (2x2x2 cm³ and 4x4x4 cm³), the test demonstrated consistent degradation profiles, confirming its scalability and robustness. Results showed rapid degradation in CEM I, moderate deterioration in CEM V, and exceptional stability in CAC. Across all binders, altered zone thicknesses obtained during these accelerated tests closely matched those observed after 49-month field exposures in real sewer conditions, yielding acceleration factors ranging from 5 to 10, depending on binder type and H₂S concentration. This study confirms the predictive ability of the proposed protocol and emphasizes the combined influence of binder composition and microstructural form (mortar vs. paste) on biogenic sulfuric acid resistance. It provides a reliable and time-efficient tool for material selection and durability assessment in wastewater infrastructure design.

## Highlights

* Accelerated test reliably replicates *in situ* degradation in sewer conditions

* Accelerated test replicates 49-month sewer degradation profiles in only 180 days

* µ-Raman coupled with chemometrics reveals zonation and confirms precise mineral changes
* Degradation is more advanced in mortar than in cement paste sections

**Keywords**

Sewerage Network, Accelerated Biodeterioration, H₂S, High-Resolution Microanalysis, Chemometrics

**1. Introduction**

Reliable sanitation systems are essential for public health and environmental protection. With current rapid urban expansion, wastewater infrastructure faces increasing pressure, while the demand for new networks continues to grow. However, cementitious materials in these environments are exposed to aggressive conditions, and their limited accessibility complicates maintenance and repair efforts. The financial burden is considerable, with rehabilitation costs estimated at €1 billion in France, €450 million in Germany, £85 million in the UK, and nearly $390 billion projected in the United States over the next 20 years [1–3]. Beyond these economic considerations, sewer failures pose severe environmental risks, emphasizing the need for durable construction materials.

One of the most damaging deterioration processes is microbially induced deterioration, driven by the biological oxidation of hydrogen sulfide (H₂S). This phenomenon has been recognized for decades [4, 5], and is now well described at a macroscopic level [2, 3, 6–9]. It arises when sulfate-reducing bacteria (SRB) generate H₂S in anaerobic zones, which then degasses and dissolves on moist pipe walls. In the aerobic zones, neutrophilic and subsequently acidophilic sulfur-oxidizing bacteria convert H₂S into sulfuric acid. The acid dissolves cement hydrates (CH, C-S-H), while secondary sulfate phases such as gypsum and ettringite form, leading to cracking and loss of structural integrity.

Assessing cementitious materials under aggressive environmental conditions remains challenging. Field tests provide realistic insights but require years of exposure and are subject to strong variability between sites, with fluctuating parameters such as H₂S concentrations, CO₂ levels, humidity, and microbial activity. Several long-term *in situ* studies have reported these differences and their impact on deterioration kinetics [8–16]. Accelerated laboratory tests offer an attractive alternative by providing controlled and reproducible conditions, but their predictive relevance depends on their ability to reproduce both the chemical and biological mechanisms of attack.

Several laboratory testing methods have been developed to evaluate cementitious materials' durability against biogenic sulfuric acid attack. During the 1980s–1990s, Bock, Sand and their collaborators pioneered microbiological simulation systems that reproduced the biogenic corrosion of concrete under controlled laboratory conditions [17–19]. These seminal studies
demonstrated the microbial origin of sewer corrosion and established the experimental framework for subsequent generations of accelerated laboratory tests. These testing methods can be broadly classified into chemical and biological approaches [20]. Chemical tests typically involve immersing specimens into synthetic acidic solutions, often sulfuric acid (H₂SO₄), to simulate deterioration. Standardized protocols, such as the German DIN 19573:2016–03, define procedures for evaluating acid resistance, with adaptations for alternative cement formulations [21, 22]. While these methods effectively reproduce acid attack, they fail to replicate the microbial biofilm interactions that drive *in situ* biodeterioration. To overcome these limitations, microbiological simulation tests integrate sulfur-oxidizing bacteria (SOB) to simulate biogenic sulfuric acid production, thereby providing a more realistic representation of microbially induced deterioration processes. Unlike purely chemical approaches, these tests account for both the microbiological activity and associated chemical reactions, offering a more comprehensive durability assessment for cementitious materials in sewer environments.

Despite these advances, the predicted ranking of materials in laboratory tests often diverges from field observations. Most studies rely primarily on macroscopic indicators such as mass loss, surface pH, and mechanical strength, which provide useful benchmarks but fail to capture the underlying microstructural changes occurring during biodeterioration. Previous investigations in this field have typically adopted this macroscopic approach, evaluating deterioration based on bulk measurements without resolving the mineralogical transformations at the microscale [20, 23–30]. Recent studies by Ayoub *et al*. emphasized the importance of employing high-precision analytical techniques, including micro-Raman mapping, to investigate cementitious material degradation [31, 32]. Their work revealed the presence of early weathering layers forming before visible deterioration appears, emphasizing the need for microscale analyses to detect subtle mineralogical and chemical changes that conventional macroscopic methods fail to identify.

A critical gap therefore remains: the durability differences between mortar and cement paste, despite their shared binder chemistry, are poorly understood, and none of the existing accelerated tests has yet quantitatively reproduced the kinetics of long-term *in situ* degradation. Until this knowledge is established, reliable material selection for sewer environments will remain uncertain. In the present study, cementitious materials based on three binders (CEM I, CEM V/A, CAC) were exposed to controlled microbial and H₂S conditions in a laboratory chamber reproducing and accelerating biogenic sulfuric acid attack. Multi-scale characterization was conducted using macroscopic monitoring, SEM-EDS, and µ-Raman spectroscopy coupled with chemometric analysis (PCA, MCR-ALS). The findings establish a
reliable and scalable protocol for predicting long-term sewer durability and demonstrate the decisive influence of both binder composition and microstructural form (mortar vs. paste).

## 2. Materials and methods

### 2.1. Characterization of samples

Cubic cementitious specimens (2x2x2 cm³) were prepared using three cement types: ordinary Portland cement (CEM I), slag and fly ash composite cement (CEM V/A), and calcium aluminate cement (CAC). For each formulation, 12 specimens were cast. To obtain composite samples combining both mortar and cement paste, a two-layer casting approach was used: a first layer of mortar (water-to-cement ratio, W/C = 0.4, and sand-to-cement ratio, S/C = 3, using standardized silica sand according to EN 196-1 [33]) was poured into the mold, followed by a second layer of cement paste (same W/C ratio, without sand). This configuration allowed direct comparison of biodeterioration kinetics between the two phases within a single specimen.

All specimens used in the biodeterioration tests consisted of this two-layer composite configuration (mortar + paste). No separate paste-only or mortar-only specimens were produced. This clarification is important as all macroscopic, microscopic, and spectroscopic analyses presented in the Results section were performed on these composite samples. The chemical composition of the various types of cement used is detailed in Table 1.

Additionally, one larger cubic specimen (4x4x4 cm³) was prepared per formulation and kept until the end of the test period, to allow access to the degraded cross-section. This was particularly necessary for CEM I, as the smaller 2x2x2 cm³ specimens showed complete chemical deterioration after six months of exposure, preventing detailed post-exposure analysis. To ensure reproducibility, specimen preparation followed standardized protocols. After casting, specimens were cured at 100% relative humidity for 24 h, followed by de-molding and storage in sealed plastic bags for 28 days (endogenous curing) before exposure to the test conditions.

<table><caption>Table 1: Chemical composition (wt. %) of the cements used in this study.</caption><thead><tr><th></th><th>Al<sub>2</sub>O<sub>3</sub></th><th>CaO</th><th>SiO<sub>2</sub></th><th>Fe<sub>2</sub>O<sub>3</sub></th><th>MgO</th><th>TiO<sub>2</sub></th><th>SO<sub>3</sub></th><th>K<sub>2</sub>O</th><th>Na<sub>2</sub>O</th><th>P<sub>2</sub>O<sub>5</sub></th><th>LOI</th></tr></thead><tbody><tr><td>CEM I</td><td>5.5</td><td>65.6</td><td>20.0</td><td>2.8</td><td>1.2</td><td>0.3</td><td>4.0</td><td>0.3</td><td>0.1</td><td>0.4</td><td>-</td></tr><tr><td>CEM V/A</td><td>10.0</td><td>45.4</td><td>31.6</td><td>4.0</td><td>2.5</td><td>0.5</td><td>0.2</td><td>1.3</td><td>0.4</td><td>0.4</td><td>-</td></tr><tr><td>CAC</td><td>51.6</td><td>38.3</td><td>5.6</td><td>1.7</td><td>0.5</td><td>2.2</td><td>0.0</td><td>0.4</td><td>0.1</td><td>0.1</td><td>-</td></tr></tbody></table>

LOI: Loss On Ignition.

Moreover, water-accessible porosity was measured on the cubic specimens using a method adapted from the French standard NF P18-459. Since this standard does not explicitly apply to mortar or cement paste samples, only the volume of the tested specimens was adjusted. The test
involved a 4 h vacuum phase at pressures below 25 mbar, followed by a 44 h water saturation period using tap water. At the end of the 48 h cycle, two measurements were taken: the hydrostatic mass ($M_{eau}$) and the saturated mass in air ($M_{air}$). The samples were then dried in a ventilated oven at 105 °C until reaching constant mass (mass variation between two weighings below 0.05%). Results are expressed both as water-accessible porosity (%) and bulk density (g/cm³) and are shown in Table 2.

<table><caption>Table 2: Water-accessible porosity and bulk density of the cement paste and mortar phases for each cement formulation. Each value represents the average ± standard deviation of 5 specimens.</caption><thead><tr><th>Cement type</th><th>Phase</th><th>Water-accessible porosity (%)</th><th>Bulk density (g/cm³)</th></tr></thead><tbody><tr><td rowspan="2">CEM I</td><td>Cement paste</td><td>38.5 ± 0.50</td><td>1.70 ± 0.02</td></tr><tr><td>Mortar</td><td>19.3 ± 0.48</td><td>2.09 ± 0.02</td></tr><tr><td rowspan="2">CEM V</td><td>Cement paste</td><td>39.5 ± 0.50</td><td>1.64 ± 0.01</td></tr><tr><td>Mortar</td><td>19.1 ± 0.54</td><td>2.13 ± 0.01</td></tr><tr><td rowspan="2">CAC</td><td>Cement paste</td><td>30.0 ± 0.46</td><td>1.86 ± 0.02</td></tr><tr><td>Mortar</td><td>15.8 ± 0.29</td><td>2.23 ± 0.01</td></tr></tbody></table>

## 2.2. Experimental protocol

Biodeterioration tests were conducted in a sealed experimental chamber, specifically designed to simulate real sewer conditions, as depicted in Figure 1. The experimental setup and exposure parameters followed the protocol established within the frame of the French national project Perfdub [34]. This protocol was developed to standardize the evaluation of the long-term performance and durability of concrete structures under aggressive environmental conditions, including biogenic sulfuric acid attack. It defines the exposure conditions, monitoring procedures, and performance indicators used in the present study.
<figure><img src="image_4.png" /><figcaption>Figure 1: Schematic representation of the experimental setup.</figcaption></figure>

### 2.2.1. H₂S production

H₂S was continuously generated by mixing a sodium sulfide (Na₂S) solution (0.25 mol.L⁻¹) with hydrochloric acid (HCl, 0.7 mol.L⁻¹) using a peristaltic pump. This reaction maintained a stable H₂S-enriched atmosphere, ensuring continuous exposure of the cementitious specimens. To prevent excessive gas accumulation, excess H₂S was neutralized at the chamber outlet by passing it through a 0.18 mol.L⁻¹ zinc acetate solution, precipitating zinc sulfide. To maintain stable reaction conditions, both the hydrochloric acid and zinc acetate solutions were renewed every 3 days throughout the experiment.

### 2.2.2. Sample pretreatment

Prior to microbial exposure, all cementitious specimens underwent a two-week abiotic pretreatment in an H₂S-enriched environment (100 ppm). This step was essential to reduce the initially high pH in the proximity of the surface, from approximately pH 12 to pH 8, thus creating favorable conditions for microbial colonization. For clarity, this parameter will hereafter be referred to as “surface pH”, acknowledging that it represents the pH measured in the aqueous layer near the specimen surface. In addition to reducing the pH, this pretreatment phase also contributed to conditioning the surface of the materials and enriching it with sulfur nutrients, made available in different forms depending on the cement type, to support microbial activity during the subsequent exposure phase.

### 2.2.3. Microbial inoculation, growth conditions, and experimental monitoring

Following pretreatment, activated sludge collected from a wastewater treatment plant in the Paris region (France) served as the microbial inoculum. The bacterial suspension was
centrifuged to concentrate the biomass, and the resulting pellet was manually and evenly spread onto the sample surfaces using a brush, ensuring homogeneous inoculation across all specimens.

To sustain microbial activity, a nutrient solution containing CaCl₂·2H₂O (0.25 g/L), NH₄Cl (1.4 g/L), MgCl₂·6H₂O (0.4 g/L), and Na₂S₂O₃·5H₂O (5 g/L) was regularly sprayed onto the samples for seven seconds every four hours via nozzles connected to a peristaltic pump controlled by a programmable controller.

Throughout the six-month exposure period, the H₂S concentration was maintained above 50 ppm, while temperature and humidity were controlled at 30°C and 100% relative humidity, respectively. These conditions were maintained using a heating belt wrapped around the chamber's water-filled base.

H₂S concentration was continuously measured using an OdaLog® sensor. A summary of the H₂S concentration data recorded over the entire exposure period is presented in Table A-1 of the Appendix.

## 2.3. Analytical methods

### 2.3.1. Macroscopic characterization

Every two weeks, a macroscopic characterization was performed to document the progressive degradation of the specimens:

- Surface pH was measured using pH test strips (accuracy ±0.2), primarily to verify that the accelerated degradation protocol was functioning consistently across all specimens, while also providing preliminary insights into chemical changes at the material surface.

- Weight and dimensions were recorded using a precision scale (±0.01 g) and a caliper (±0.02 mm), respectively, to monitor mass and dimensional variations over time.

- Photographs were systematically taken to visually track surface alterations and degradation patterns. Photographs were systematically taken to visually track surface alterations, such as discoloration, cracking, spalling, and the appearance of sulfate deposits, in order to document the progression of biodegradation over time.

### 2.3.2. Mineralogical and chemical characterization

To better understand the chemical and mineralogical evolution of the specimens, detailed analytical techniques were applied. One sample per formulation was extracted at each of the three selected time points—T0 (end of pretreatment), T90 (mid-exposure), and T180 (end of the test period)—then embedded in epoxy resin, sectioned, and polished with diamond abrasives in ethanol to the micron scale, ensuring optimal surface quality for examination.
Cross-sections were first observed using a digital optical microscope (KEYENCE, ×30 magnification) to assess macroscopic integrity and identify the boundaries of degraded regions.
For the calculation of penetration depths, ImageJ software was used.

**a- SEM-EDS analysis**

Polished cross-sections were analyzed using scanning electron microscopy (SEM, Quanta 400 FEI), coupled with energy-dispersive X-ray spectroscopy (EDS, Xplore30, Oxford), to investigate chemical changes induced by biodeterioration in depth.

Before analysis, specimens were carbon-coated to improve conductivity. Backscattered electron (BSE) imaging provided a panoramic view of degradation zones for microstructure observation, while EDS mapping (operated at 20 kV) quantified elemental variations across the whole section. The Kα-ray intensities of key elements present in cement (S, Si, Ca, Mg, K, Al, Fe) were measured to assess compositional modifications resulting from biogenic sulfuric acid attack following H₂S exposure.

**b- Micro-Raman mapping and chemometric analysis**

Raman spectroscopy analyses were performed using an HR800 LabRam spectrometer (Horiba Scientific, Jobin-Yvon division) equipped with a 514 nm Argon-ion laser (Coherent). Despite potential fluorescence interference, this wavelength is widely used for spectroscopic cementitious material analysis [35–37, 31, 32].

The system was coupled with an Olympus BX microscope, employing an MPlanN ×50 (NA 0.75) objective, yielding a diffraction-limited spot size of ~1 µm. Two-dimensional spectral mappings were acquired using a motorized XY stage, with step sizes ranging from 150 to 250 µm, and an integrated Z autofocus function for precise focusing at each point.

Depending on the region of interest, Raman maps covered areas ranging from a minimum 3.5 x 5.5 mm to larger regions up to 10 x 10 mm of the cross-section. These acquisition parameters enabled the coverage of relatively large surface areas while maintaining sufficient spatial resolution for mineralogical discrimination.

Spectral acquisition was performed with an 1800 grooves/mm grating and an Ultra-Low Frequency (ULF) module, achieving a spectral resolution of ~2 cm⁻¹ over a range of 40–1800 cm⁻¹. The laser power was set to ~1 mW at the sample surface, with an integration time of 5 s per spectrum and two accumulations per acquisition to improve the signal-to-noise ratio. The instrument was controlled via LabSpec 6 software.

Regions selected for mapping were systematically identified through optical microscopy (Keyence, reflected light), focusing on the most visibly degraded zones of the cross-sections.
For each specimen, multiple independent maps were collected on different regions (paste,
mortar, and mixed zones) to ensure that the mineralogical trends observed were representative of the overall deterioration pattern.

Raman spectra provide extensive data on the chemical and physical composition of cementitious phases. However, due to signal overlap and spectral complexity, conventional visual inspection is often insufficient. Chemometric techniques offer a powerful and objective approach to extracting meaningful chemical information from large spectral datasets, particularly in systems with closely similar spectral features [38]. These methods have been increasingly applied in the analysis of civil engineering materials, particularly in Raman spectroscopy [36, 39, 31, 40].

To analyze the degradation processes in cementitious materials, two chemometric approaches were employed:

- Principal Component Analysis (PCA) for exploratory data analysis and dimensionality reduction.

- Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) for signal decomposition and phase identification.

Before analysis, data preprocessing was performed to enhance spectral quality and minimize interferences [41]. This included spike removal, baseline correction (Whittaker filter), L1-norm normalization, and mean centering for PCA [42]. Mean centering was not applied for MCR-ALS, as this method is designed to separate pure spectral contributions [43].

PCA transforms high-dimensional spectral data into a set of uncorrelated orthogonal axes called Principal Components (PCs), capturing the main sources of variance in the dataset [44, 45]. The first PC (PC1) represents the dominant variance, while subsequent PCs describe additional, uncorrelated residual variability. This approach facilitates the visualization of patterns, clustering, and outliers within the dataset [38]. However, PCA does not provide direct spectral interpretations, as its components can contain negative values, which are not chemically meaningful [43, 46].

To overcome this limitation, MCR-ALS was applied to unmix spectral signals and retrieve the pure spectra of distinct mineralogical phases [47]. This method assumes a bilinear additive model, where each spectral component represents a unique phase, allowing the reconstruction of chemically accurate spectral profiles and spatial distribution maps of the mineralogical phases [48]. To ensure physically meaningful results, constraints such as non-negativity were imposed [49].
All data processing and chemometric analyses were conducted in MATLAB® R2023a (MathWorks, USA) using PLS Toolbox version 9.2.1 (Eigenvector Research, Inc.) for PCA and MCR-ALS Toolbox version 2.0 for MCR-ALS analysis.

# 3. Results

## 3.1. Macroscopic Evolution of Cementitious Specimens Under H₂S Exposure

The macroscopic degradation of the cement specimens was monitored over time through variations in weight, surface pH, and dimensions, as well as through visual observation. These indicators provide an initial assessment of the deterioration process and enable comparative analysis between the studied cement formulations (CEM I, CEM V and CAC).

### 3.1.1. Surface pH Evolution

To ensure the proper functioning of the accelerated degradation protocol and monitor the acidification kinetics, surface pH measurements were performed every 15 days throughout the 180-day exposure period. All measurements were taken on the mortar section (upper part) of each specimen, where surface reactions are expected to be more representative of the real-world infrastructure elements. The evolution of surface pH is presented in Figure 2-a.

All specimens showed a clear and significant decrease in surface pH over time, confirming that the biogenic sulfuric acid attack was successfully initiated and sustained across all cement formulations.

* CEM I exhibited a continuous and sharp pH drop, from initial values around 10 down to approximately 1.5 by day 180, indicating intense acidification and limited buffering capacity. Notably, the pH continued to decrease steadily beyond 160 days, showing that the degradation process remained active and severe until the end of the test.

* CEM V showed a more moderate decline, with surface pH stabilizing around 5 after 160 days, indicating partial resistance to acidification, likely due to its blended composition and reduced portlandite content. This behavior reflects improved acid resistance compared to CEM I, although significant acidification still occurred.

* CAC also stabilized at pH 5, but with a less steep decline overall, reflecting a higher inherent resistance to acidification and microbial activity.

These results confirm that the accelerated test protocol effectively simulates the sulfuric acid attack process. They also highlight the influence of cement composition on biogenic acid resistance, with CEM I being the most vulnerable, followed by CEM V, while CAC exhibits the most robust performance. The stabilization of surface pH for both CEM V and CAC after 160 days suggests a limit to acid penetration or a chemical equilibrium reached at the surface, unlike CEM I, which continued degrading rapidly.
<figure><img src="image_5.png" /><figcaption>Figure 2: Average macroscopic evolutions of the specimens over 180 days: (a) surface pH variation, (b) weight change (%), and (c) dimensional change (%). Error bars indicate the standard deviation (n=12).</figcaption></figure>

### 3.1.2. Visual Evolution of Surface Degradation

The photographic documentation of CEM I, CEM V, and CAC specimens over 180 days of exposure in an H₂S-enriched microbial environment is presented in Figure 3. Initially, all specimens exhibited smooth and uniform surfaces, reflecting their prepared state. However, progressive surface degradation became evident over time, with differences in both timing and severity across the three cementitious materials and between the mortar (upper part) and cement paste (lower part) sections of each specimen.

- CEM I specimens exhibited early signs of deterioration, with discoloration clearly visible from the very first sampling point (15 days). This was followed by rapid surface roughening, visible cracking, and the formation of white sulfate deposits, likely corresponding to gypsum precipitation. The damage intensified progressively, resulting in large-scale spalling and, by day 165, near-complete structural disintegration. These observations confirm the high vulnerability of CEM I to biogenic sulfuric acid attack, due to its chemical composition and high porosity (Table 1 and 2).

- CEM V specimens showed initial discoloration starting around day 30, followed by the appearance of surface cracking and localized spalling of the outer layer. While degradation progressed with time, the overall structural integrity of the specimens remained more preserved than for CEM I.

- CAC specimens exhibited initial discoloration around day 30; however, no significant cracking or spalling was observed even after 180 days of exposure. Aside from minor surface irregularities and gradual discoloration, the material largely preserved its structural integrity. Localized degradation zones were identified, likely associated with the dissolution of calcium aluminate hydrates under acidic conditions. The enhanced
resistance of CAC is attributed to its mineralogical composition and low porosity, particularly in the mortar matrix.

In all specimens, the mortar section (top) appeared more affected than the cement paste section (bottom). Despite the fact that porosity was systematically higher in the cement paste than in the mortar phase for all formulations (Table 2), the mortar regions exhibited more pronounced visual degradation. It is also important to note that the images shown at 180 days in Figure 3 correspond to the larger 4x4x4 cm³ cubic specimens.

<figure><img src="image_6.png" /><figcaption>Figure 3: Evolution of the visual appearance of specimens (initial size: 2x2x2 cm³) exposed in the H₂S chamber, from pretreatment to the end of the test. The 180-day images correspond to the larger 4x4x4 cm³ specimens. For each specimen, the mortar section is located at the top and the cement paste section at the bottom.</figcaption></figure>

### 3.1.3. Weight and Dimensional Variations

Weight and dimensional measurements were performed every 15 days to monitor the evolution of material behavior under biogenic sulfuric acid exposure. The results, shown in Figure 2-b,c were recorded on the mortar sections of the specimens, where degradation was generally more pronounced.

During the first 110 days, all cement formulations exhibited a slight increase in weight, corresponding to water ingress under the constant 100% relative humidity conditions in the test chamber. This behavior, commonly reported in the literature [8, 9, 27, 50], reflects saturation
of the pore network; more pronounced in mortars due to their higher pore connectivity and
presence of the interfacial transition zones (ITZ).

However, beyond 120 days, degradation-dependent mass and dimensional variations diverged
significantly among the three cements:

* CEM I specimens exhibited a sharp increase in mass starting from day 100, reaching up to 33% average weight gain by day 160, followed by a decrease to ~27% by day 190. This pronounced weight gain is indicative of significant material alteration, likely due to the formation of expansive products such as gypsum and ettringite. These transformations are consistent with *in situ* findings [7, 11, 12, 13, 50], and visually confirmed by the complete structural disintegration observed (Figure 3). The associated swelling (~14%) reinforces the hypothesis of internal stress induced by expansive phase formation.

* CEM V specimens showed a more gradual and moderate weight increase, reaching ~18% by day 190. Dimensional changes remained limited until day 160, after which a swelling of ~7% was noted. This delayed swelling suggests a slower but still progressive sulfate ingress and reaction, leading to the formation of expansive products over time, albeit less aggressively than in CEM I.

* CAC specimens, in contrast, demonstrated minimal weight change throughout the entire exposure period, remaining below 5% from day 15 to day 190. No significant dimensional changes were observed beyond the initial water uptake. This confirms CAC's high resistance to sulfate-rich acidic environments, in agreement with previous studies highlighting its limited formation of expansive sulfate phases under such conditions [9, 11, 24, 50, 51].

Interestingly, the cement paste sections exhibited significantly less swelling than their mortar counterparts.

These macroscopic results confirm that:

* CEM I is highly vulnerable to microbial sulfate attack, with early onset and severe degradation;

* CEM V demonstrates intermediate resistance, showing delayed deterioration and more moderate expansion;

* CAC exhibits excellent dimensional and chemical stability, with negligible alteration.
These findings corroborate the trends observed in surface pH and visual inspections, and
provide the basis for the microstructural and mineralogical analyses presented in the following
sections.

**3.2. Microstructural and Chemical Evolution**

To further investigate degradation mechanisms at the microstructural level, a SEM-EDS
analysis was performed on the entire polished cross-sections of CEM I, CEM V, and CAC
specimens exposed to the H2S-enriched microbial environment at three distinct time points: T0
(end of the pre-treatment period), 90 days (mid-exposure), and 180 days (end of the test period).
This analysis provided detailed insights into morphological alterations, chemical composition
changes, and element migration patterns associated with biogenic sulfuric acid attack.
Additionally, porosity and degradation profiles were established through elemental mapping,
allowing for a comprehensive assessment of sulfate penetration and depth distribution of key
elements within the cementitious matrix. These findings allowed for the identification of
progressive deterioration patterns and contributed to a better understanding of the
microstructural stability of each cement formulation under prolonged exposure to H2S and
microbial activity.

**3.2.1. Sulfur Penetration in Specimens from the Pre-Treatment Phase**

The elemental sulfur distribution after the pre-treatment phase (15 days of exposure to 100 ppm H2S in the absence of bacterial activity) is presented in Figure 4. Although no microbial oxidation occurred at this stage, the results indicate significant sulfur diffusion into the specimens, visible as lighter blue areas. This phenomenon is purely physical and driven by the permeability and pore network characteristics of the cementitious materials, not by any chemical degradation.

The extent of sulfur ingress was strongly correlated with the material's water-accessible porosity (Table 2). Portland cement-based formulations (CEM I and CEM V, Figure 4-a,b) showed notably higher sulfur diffusion compared to calcium aluminate cement (CAC, Figure 4-c), due to their more interconnected and accessible pore networks. For example, in the mortar part of the specimens, a visual analysis highlights greater and deeper sulfur penetration in CEM I (Figure 4-a), with an average penetration depth of 2.34 mm, reflecting its relatively high water porosity of nearly 19.3%. This sulfur diffusion suggests low chemical resistance and a highly porous microstructure, which facilitates biodeterioration mechanisms.

In contrast, CAC (Figure 4-c) exhibited the highest resistance to sulfur penetration, with an average depth of only 0.68 mm, corresponding to its relatively low water-accessible porosity of 15.8%. This reduced porosity likely limits the diffusion of sulfur species and the extent of
chemical interactions, contributing to the superior durability of CAC against biogenic sulfuric acid attack.

Interestingly, sulfur diffusion was more pronounced in the mortar sections (upper regions of the specimens) than in the cement paste sections (lower regions). In CEM I, for instance, the average sulfur penetration depth was 2.34 mm in the mortar versus 1.30 mm in the cement paste. In CEM V, this trend was similar: 2.42 mm in the mortar and 1.29 mm in the cement paste. Even in CAC specimens, the same pattern was observed (0.68 mm in the mortar vs. 0.37 mm in the paste).

Several factors may explain the greater diffusion observed in mortar sections:

* Lower cement paste volume in mortar reduces the amount of dense matrix available to limit gas penetration. Since H₂S diffusion predominantly follows the cementitious matrix, it tends to bypass the less porous aggregates, favoring matrix-rich zones.

* Greater pore connectivity in mortar, particularly at the interfacial transition zones (ITZ) between the aggregates and the paste, offers preferential pathways for gas migration. These ITZs are known to present higher porosity and larger capillary pore entrances, sometimes exceeding several microns, which can facilitate both H₂S diffusion and, later, microbial colonization [52, 53].

* Microstructural heterogeneity and microcracks may further enhance gas ingress by creating continuous paths through the material.

These findings underscore the importance of microstructural features, especially porosity and ITZ characteristics, in governing the early physical diffusion of H₂S. Although no visible chemical transformation was observed at this stage, this initial ingress is a critical precursor that sets the stage for subsequent microbial activity and acid generation in the later phases of exposure.
<figure><img src="image_7.png" /><figcaption>Figure 4: Elemental sulfur distribution obtained by SEM-EDS after pretreatment of cementitious samples in a 100 ppm-H₂S enriched atmosphere: cross-section analysis of (a) CEM I, (b) CEM V, and (c) CAC. (Lighter blue indicates sulfur penetration)</figcaption></figure>

### 3.2.2. Evolution of Elemental Distribution at 90 and 180 Days of Exposure

SEM-EDS analysis, combined with elemental mapping, enabled a detailed investigation of the elemental distribution and degradation progression within the specimens over time. The results revealed clear zonation patterns, which differed between Portland cement-based specimens (CEM I and CEM V) and calcium aluminate cement (CAC) specimens. These zones were visually distinguishable in backscattered electron (BSE) imaging, showing microtextural contrasts and variations in elemental intensity across the cross-sections.

#### Portland cement-based specimens (CEM I and CEM V)

For both CEM I and CEM V specimens, four distinct degradation zones were systematically observed across the 90- and 180-day specimens, as illustrated in Figure 5-a, b.

* Zone 1 – Surface enrichment layer:

This outermost layer, clearly distinguishable by its dense microstructure, showed strong enrichment in sulfur (S) and calcium (Ca), consistent with the formation of sulfate minerals. This reaction front corresponds macroscopically to the observed mass increase due to mineral precipitation and dimensional swelling linked to crystallization pressure. This layer effectively acts as a "reaction crust" developing from surface condensation and sustained sulfuric acid attack.

* Zone 2 – Highly porous transition zone:

Beneath the surface, this chemically depleted zone displayed marked porosity and was strongly enriched in silicon (Si), with near-total leaching of Ca and S. The matrix appeared highly eroded, reflecting binder dissolution and paste disintegration. This zone corresponds to the loss of mechanical cohesion and weakening observed
macroscopically, especially in CEM I, as evidenced by early cracking and spalling (Section 3.1.2).

* **Zone 3 – Intermediate alteration zone:**
  Further inward, an accumulation of aluminum (Al), iron (Fe), and magnesium (Mg) was detected, accompanied by delayed sulfur and calcium re-incorporation. This region likely reflects the formation of secondary phases such as aluminum- or iron-based sulfates and the progressive diffusion of aggressive species. Its presence demonstrates the gradual inward propagation of the degradation front.

* **Zone 4 – Pristine Zone (unaltered matrix):**
  The innermost region retained its original microstructure and composition, indicating no significant chemical alteration at this stage. This preserved zone is more prominent in CEM V than in CEM I, in agreement with the macroscopic stability observed for CEM V (Section 3.1).

At 180 days, small CEM I specimens (2x2x2 cm³) showed complete degradation, with the pristine zone fully absent, confirming total chemical deterioration. This was reflected by the sharp pH drop and severe dimensional changes (Section 3.1). By contrast, the degradation front in CEM V remained more superficial even after 180 days, with the pristine core still preserved. This indicates that the composite nature of CEM V delays the progression of the aggressive front, reinforcing its superior performance over CEM I under biogenic sulfuric acid attack.

To extend and validate these findings, larger specimens (4x4x4 cm³) were also analyzed at 180 days under identical biogenic conditions. The aim was to assess whether the degradation mechanisms identified in the smaller 2x2x2 cm³ specimens remained consistent at a larger scale. These larger CEM I and CEM V samples exhibited similar four-zone degradation profiles (Figure 6), confirming that the observed zonation is independent of scale; however, the thicknesses of the individual zones differed. In CEM I, the larger sample retained just enough integrity to allow microstructural analysis, yet showed deep sulfur ingress and extensive calcium depletion. CEM V, meanwhile, continued to show a more superficial degradation front and preservation of the inner matrix. These results confirm the robustness of the degradation mechanism across specimen sizes and validate the reliability of the accelerated testing protocol.

## Calcium Aluminate Cement (CAC) specimens

CAC specimens exhibited three distinct zones, with significantly less degradation than Portland cement-based materials, reflecting their greater chemical stability under prolonged biogenic sulfuric acid exposure (Figure 5-c). The zonation was consistent at both 90 and 180 days, although slightly more pronounced at the later stage.
* Zone 1 – Surface precipitation zone:

This outermost layer was not a deeply altered region but rather a superficial reaction layer, where sulfur and calcium accumulated, forming sulfate-rich phases (likely gypsum). This precipitation occurred directly at the surface, as a result of acid-base reactions with sulfuric acid, and corresponds to the limited mass gain observed macroscopically (Section 3.1.3). Unlike CEM I and CEM V, this zone does not extend deeply, suggesting a very slow ingress of aggressive species.

* Zone 2 - transition zone:

Located just beneath the surface, this layer exhibited continued sulfur presence but with a notable reduction in calcium content, alongside localized accumulation of iron (Fe) and magnesium (Mg). Interestingly, aluminum (Al) was no longer detected in this zone, suggesting partial dissolution of aluminate hydrates under acidic exposure.

* Zone 3 – Pristine core zone (unaltered matrix):

The inner part of the specimen remained chemically stable, with high concentrations of anhydrous phases and well-distributed Al, showing no evidence of sulfur ingress or calcium leaching. This zone was thicker than in Portland cements, indicating that the aggressive front advanced very slowly and did not significantly affect the microstructure.

At 180 days, the zones were slightly more developed but remained confined to the outer layers. Analysis of larger CAC specimens (4x4x4 cm³) confirmed this behavior, with limited sulfur penetration and overall negligible calcium loss, preserving the integrity of the core microstructure. These results underscore CAC's robust resistance to biogenic sulfuric acid attack, regardless of specimen size.

The restricted sulfur penetration and limited calcium depletion in CAC suggest that, unlike CEM I and CEM V, this formulation does not promote the formation of extensive sulfate-rich degradation products. This behavior may result from several interrelated factors:

- The lower initial calcium content in CAC reduces the availability of reactive species for gypsum formation.

- Although some dissolution of aluminate hydrates occurs in the altered surface layers, Al-bearing phases such as AH₃ are overall less sensitive to acid attack than the portlandite-rich phases in CEM I / CEMV.

- Potential mineralogical transformations of AH₃ into more stable polymorphs such as gibbsite or bayerite could contribute to pore refinement or partial blocking of the matrix, thereby limiting sulfur ingress.
<figure><img src="image_8.png" /></figure>

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<figure><img src="image_14.png" /><figcaption>(a) CEM I</figcaption></figure>

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<figure><img src="image_21.png" /><figcaption>(b) CEM V</figcaption></figure>

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<figure><img src="image_28.png" /><figcaption>(c) CAC</figcaption></figure>

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Figure 5: SEM-EDS Elemental Distribution of (a) CEM I, (b) CEM V, and (c) CAC Specimens after 180 Days of Exposure. The mortar section is located at the top and the cement paste section at the bottom of each cross-section.

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<figure><img src="image_32.png" /><figcaption>CEM 1</figcaption></figure>

<caption>CEM V</caption>

<caption>CAC</caption>
Figure 6: SEM-EDS elemental distribution maps of 4x4x4 cm³ specimens (CEM I, CEM V, and CAC) after 180 days of exposure to a biogenic H₂S-enriched atmosphere. The mortar section is located at the top and the cement paste section at the bottom of each cross-section.

### 3.3. Mineralogical Evolution

Raman spectroscopy and imaging are essential tools for micro-scale phase characterization of cement pastes in their anhydrous, hydrated, and carbonated states [39, 40, 54–56]. In this study, Raman mapping was performed on cross-sections of the 2x2x2 cm³ specimens at three distinct time intervals: T0 (completion of pre-treatment), 90 days (mid-exposure), and 180 days (final test stage). Multiple mapping sessions were conducted for each cross-section, some focusing exclusively on cement paste, others on mortar, and some encompassing areas containing both materials. Figure A-1 of the Appendix provides an overview of a CEM V specimen analyzed at 180 days of exposure, illustrating the selected mapping zones. The leftmost image presents a macro-scale view of the cross-section, where red rectangles highlight the regions subjected to Raman mapping. The subsequent images, captured at 20x magnification, display the corresponding mapped areas, allowing for a detailed assessment of phase transformations and degradation extent.

The primary objectives were to evaluate the impact of H₂S exposure on material composition, to assess the penetration depth of the biodeterioration front, and to determine the influence of mortar composition on these processes. Additionally, the study aimed to evaluate whether the accelerated test conditions employed in this experiment effectively reflect real sewer system environments.

For each mapped region, PCA was first employed to estimate the number of chemically distinct species present. The analysis began with an exploratory selection of 20 principal components (PCs), determined through a trial-and-error approach. Subsequently, the data were analyzed using MCR-ALS, with non-negativity constraint applied to both concentration profiles and spectral signatures. This approach enabled the isolation of spectral contributions from both intact cement phases and sulfate-related degradation products, as well as their spatial localization within the microstructure.

#### **3.3.1. Portland cement-based specimens (CEM I and CEM V)**

Raman spectroscopy combined with MCR-ALS analysis revealed similar degradation mechanisms for CEM I and CEM V specimens, albeit with marked differences in the extent and depth of mineralogical transformation over time (Figure 7 and Figure 8).

- T0 – Pre-Treatment Phase
For both cement types, Raman mapping identified early chemical alterations after the 15-day abiotic H₂S exposure. In CEM I, distinct Raman bands corresponding to gypsum (spectral band at ~1008 cm⁻¹) and traces of elemental sulfur (spectral band at ~470 cm⁻¹) were detected near the surface, indicating that calcium sulfate formation occurred even in the absence of microbial activity; highlighting the high chemical reactivity and vulnerability of CEM I. In contrast, CEM V exhibited only traces of elemental and no detectable gypsum, suggesting a reduced reactivity under identical conditions, likely due to its blended formulation and lower portlandite content.

- 90 Days – Mid-Exposure

At this stage, both specimens exhibited mineralogical signatures characteristic of sulfur and gypsum, but the extent of degradation varied substantially. CEM I showed widespread gypsum formation penetrating both mortar and cement paste, with a deeper degradation front in the mortar, likely due to its higher effective porosity. For CEM V, gypsum was also identified but was restricted to a much thinner superficial zone. The rest of the section remained largely unaltered, with a substantial portion of the inner matrix still exhibiting spectral features of intact hydration phases. These zones were labeled in the spatial distribution maps of Figure 7 and Figure 8 as “intact matrix”, given the absence of strong evidence linking them to degradation products in this context.

- 180 Days – Final Stage

By 180 days, CEM I exhibited complete mineralogical transformation, as Raman spectra were entirely dominated by gypsum with no remaining spectral features of unaltered cement phases. This observation confirms a total structural and chemical breakdown of the matrix under biogenic sulfuric acid attack. In contrast, CEM V showed only a moderate progression in sulfate-induced transformation. Gypsum remained confined to the outermost region, and a large portion of the cement paste and mortar still retained spectral features of intact hydration products. This confirms the greater durability of CEM V under prolonged exposure, likely due to the dilution of reactive phases and the lower availability of free lime for sulfate reaction.

These observations emphasize the critical influence of binder composition on the kinetics and depth of biodeterioration. The gypsum-rich layer was significantly thicker in CEM I, correlating with its greater mass loss and swelling (cf. Section 3.1), while the preserved inner matrix in CEM V highlights its better resistance to sulfate-induced degradation. The presence of an identifiable “intact matrix” zone in CEM V after 180 days reinforces the protective effect of blended cements against aggressive biogenic environments.
<figure><img src="image_33.png" /><figcaption>Figure 7: Mineralogical evolution of CEM I specimens under biogenic sulfuric acid exposure. (a) Macro-scale cross-sections with red rectangles indicating the mapped zones. (b) Optical images of cement paste and mortar sections at T0, 90 days, and 180 days. (c) MCR-ALS spatial distribution maps of degradation phases and intact matrix.</figcaption></figure>
<figure><img src="image_34.png" /><figcaption>Figure 8: Mineralogical evolution of CEM V specimens under biogenic sulfuric acid exposure. (a) Macro-scale cross-sections with red rectangles indicating the mapped zones. (b) Optical images of cement paste and mortar sections at T0, 90 days, and 180 days. (c) MCR-ALS spatial distribution maps of degradation phases and intact matrix.</figcaption></figure>

### 3.3.2. Calcium Aluminate Cement (CAC) specimens

- T0 – Pre-Treatment Phase:

At T0, Raman mapping of CAC specimens showed a uniform mineralogical structure. MCR-ALS identified well-defined calcium aluminate (CA, CaAl₂O₄) signatures across the section. The spatial distribution confirmed the structural integrity of both the cement paste and the mortar, with no detectable degradation products or signs of chemical transformation (Figure 9). This observation is consistent with SEM-EDS results showing limited sulfur presence at this
stage, highlighting the initial stability of CAC under short-term H₂S exposure in the absence of microbial activity.

- 90 Days – Mid-Exposure:

By 90 days of exposure, Raman mapping revealed the early onset of sulfate attack. The first mapped region, which included both cement paste and mortar, still exhibited the intact CA phase but also revealed localized formation of ettringite (spectral band at 985 cm⁻¹), particularly near the exposed surface. This suggests initial chemical transformation of hydrated aluminate phases under the influence of biogenic sulfuric acid.

In the mortar portion, while CA was still evident (Figure 9), its distribution was neither homogeneous nor fully continuous, indicating that the zone was partially altered. The red line in Figure 9 marks the boundary between the fully altered outer layer and the partially altered zone, where residual CA remains detectable. This is further supported by the spatial distribution of ettringite (Figure 10): a first altered zone near the surface shows high ettringite intensity, followed by a more diffuse and less ettringite content.

In a second mapped region consisting solely of cement paste, MCR-ALS identified CA, ettringite, and elemental sulfur (spectral band at 470 cm⁻¹), confirming microbial activity and the beginning of a sulfur cycle. The sulfur was concentrated in a thin superficial layer, preceding the ettringite zone, suggesting a degradation front initiated by sulfur oxidation before sulfate crystallization. Although the extent of these degradation products was still limited compared to Portland cement specimens, their presence confirmed that CAC, while more resistant, was not entirely immune to biodeterioration under sustained exposure.

- 180 Days – Final Stage:

At the end of the exposure duration, more extensive mineralogical alteration was observed, particularly in mortar. Raman mapping of both the cement paste and the mortar revealed the presence of elemental sulfur, ettringite, and gypsum as degradation products, concentrated in the outermost layers (Figure 10). The CA phase remained detectable in deeper areas but was reduced in intensity compared to earlier stages. The degradation front was clearly stratified: sulfur near the surface, followed by gypsum and then ettringite. Cement paste followed a similar pattern, though the progression remained limited (Figure 9).

These findings complement SEM-EDS observations (Section 3.2.2) and confirm that CAC exhibits superior resistance to biodeterioration, with degradation confined to the outermost regions and core mineralogy preserved. The more pronounced alteration in mortar reflects its higher porosity and structural heterogeneity, promoting deeper ingress of aggressive species.
These results are consistent with previous findings and affirm the suitability of CAC for sewer environments [9, 11, 14, 27, 32].

<figure><img src="image_35.png" /><figcaption>Figure 9: Spatial distribution of CA phase in CAC specimens over time. (a) Cross-sections with red rectangles indicating the mapped regions. (b) Optical images of the analyzed sections. (c) MCR-ALS spatial maps of CA, (d) Representative Raman spectra (cm⁻¹) extracted from the CA phase at each time step.</figcaption></figure>
<figure><img src="image_36.png" /><figcaption>Figure 10: Spatial distribution of sulfate-induced degradation products in CAC specimens after 90 and 180 days of H₂S exposure. (a): Optical images of the analyzed cross-sections with annotated zones showing the presence of ettringite, sulfur, and gypsum. (b): Raman mapping results showing the spatial distribution of each phase obtained by MCR-ALS.</figcaption></figure>

Raman spectroscopy confirmed that while all cement types exhibited some degree of phase transformation under biogenic sulfuric acid exposure, the kinetics and extent of degradation varied considerably. CEM I showed early and pervasive sulfate formation; CEM V exhibited delayed, superficial damage; and CAC demonstrated minimal mineralogical alteration, with core phases largely intact. These differences highlight the critical role of binder composition and porosity in determining resistance to microbial-induced degradation in aggressive environments.

Moreover, the results obtained in this study are in good agreement with those previously reported by Ayoub *et al.* [32], where cementitious specimens (60 mm in diameter) were exposed *in situ* in real sewer environments for 49 months. In both the accelerated and *in situ* studies, Raman spectroscopy consistently identified gypsum as the dominant degradation product in CEM I and CEM V, with elemental sulfur deposits also frequently detected. Similarly, CAC
specimens in both studies exhibited only limited formation of gypsum and sulfur confined to a very thin surface layer, further confirming their superior chemical stability.

A notable difference, however, concerns the detection of bayerite and gibbsite in the *in situ* CAC specimens. These aluminum hydroxides were not observed in the present study, most likely due to a spectral acquisition limit of 1800 cm⁻¹, whereas bayerite and gibbsite exhibit Raman peaks at 3420 cm⁻¹ and 3364 cm⁻¹, respectively. This limitation prevented the full identification of secondary Al-bearing phases, which could otherwise indicate long-term mineral stabilization.

These converging results validate the mineralogical relevance and predictive capacity of the accelerated test used here. The strong consistency in mineralogical phases between short-term accelerated testing and long-term *in situ* exposure strengthens the confidence in using µ-Raman spectroscopy and chemometric analysis as reliable tools for assessing the durability of cementitious materials exposed to biogenic sulfuric acid attack.

Overall, the combined use of SEM-EDS and Raman spectroscopy enabled a multi-scale understanding of biodegradation processes in H₂S-enriched environments. The observed mineralogical and microstructural changes demonstrate a strong correlation between binder type, porosity, and durability performance. These insights are essential for guiding the selection and optimization of resistance materials for sewer infrastructure applications.

# 4. Discussion

## 4.1. Influence of cement type on biochemical resistance

The degradation behavior of the three studied cementitious formulations (CEM I, CEM V, and CAC) under biogenic sulfuric acid exposure revealed clear distinctions in durability performance. CEM I was the most vulnerable, exhibiting rapid acidification, high mass gain due to sulfate phase formation, early cracking, and complete mineralogical transformation. These phenomena are directly linked to its high portlandite content, high porosity, and chemical reactivity.

CEM V, a blended binder containing slag and fly ash, exhibited much more resistance. The lower amount of reactive portlandite and a denser hydration matrix reduced sulfate attack kinetics and limited the extent of mineralogical transformation. This improved behavior is attributed to the blended nature of the binder and its reduced portlandite content, which limits gypsum formation despite a comparable porosity in the paste phase (Table 2). While surface degradation occurred, the preservation of core material and delayed expansion confirm the beneficial role of composite binder formulations in enhancing acid resistance.
CAC displayed the best performance among the three, with negligible mass or dimensional changes, limited sulfur ingress, and preservation of core mineralogy even after 180 days. This resistance is primarily attributed to the lower calcium content, more stable hydration products (e.g., CA, $AH_3$), and reduced pore quantity, which jointly hinder aggressive ion penetration and expansive phase formation.

These findings confirm that binder composition, particularly calcium availability and the presence of stable aluminate phases, is a determining factor for resistance to biogenic sulfuric acid attack. Moreover, the consistent degradation profiles across cement types and specimen sizes demonstrate the reliability of the accelerated test method.

## Mechanistic interpretation of the degradation behavior

The differences in durability observed among CEM I, CEM V and CAC can be directly explained by the chemical pathways governing biogenic sulfuric acid attack. Once $H_2SO_4$ is produced on the concrete surface by sulfur-oxidizing bacteria, the first reaction is the rapid consumption of portlandite ($Ca(OH)_2$), which neutralizes acidity and precipitates gypsum according to:

$$Ca(OH)_2 + H_2SO_4 \rightarrow CaSO_4 \cdot 2H_2O$$

This reaction is particularly critical for CEM I, whose high portlandite content makes it highly reactive and leads to abundant gypsum formation. Once portlandite is exhausted, sulfuric acid begins decalcifying the C–S–H gel, producing additional gypsum and leaving behind a silica-rich residue:

$$C-S-H + H_2SO_4 \rightarrow SiO_2 \cdot H_2O + CaSO_4$$

In Portland-based systems, sulfate ions also react with calcium aluminate hydrates to form ettringite, another expansive sulfate phase:

$$C-A-H + 3CaSO_4 \cdot 2H_2O \rightarrow Ettringite$$

The combined precipitation of gypsum and ettringite explains the early cracking and swelling observed in CEM I specimens.

CEM V exhibits slower kinetics because its blended composition (slag + fly ash) produces a microstructure with lower portlandite content and more stable aluminosilicate hydrates. The reduced quantity of $Ca(OH)_2$ limits the rate of gypsum formation, and the denser C–S–H matrix formed by latent hydraulic reactions decreases acid penetration, slowing both neutralization and decalcification fronts.
In CAC, the mechanism of attack differs fundamentally. CAC hydrates contain much less calcium and a higher proportion of aluminum-rich phases (CAH₁₀, C₂AH₈, C₃AH₆). Under acidic attack, calcium is leached but stable aluminum hydroxide gels Al(OH)₃ precipitate:

$$Ca - Al - H + H^{+} \rightarrow Al(OH)_{3} + Ca^{2+}$$

Unlike gypsum and ettringite, Al(OH)₃ does not produce expansive stresses and forms a relatively dense layer that partially slows further acid ingress. This explains the minimal dimensional change, limited sulfur penetration, and preservation of pristine mineralogy observed even after 180 days.

Taken together, these chemical reactions clarify why CEM I undergoes rapid, expansive degradation, while CEM V shows delayed and attenuated deterioration, and CAC demonstrates superior resistance. The microstructural evolution and zonation observed in SEM-EDS and Raman analyses are consistent with the sequential depletion of alkaline phases and the nature of secondary products formed in each binder system despite the different spatial resolution of each of these analytical techniques.

Moreover, the observed degradation profiles, both morphologically and chemically, closely align with those reported under real in situ conditions in previous field studies [8, 9, 14, 31, 32, 51] without any control on H₂S concentration nor temperature or humidity. This consistency validates the experimental setup as a robust and time-efficient alternative to long-term field exposure, making it a powerful tool for evaluating and optimizing cementitious materials for sewer environments.

## 4.2. Mortar vs. Cement Paste: The Role of Microstructure

A consistent observation across all cement formulations was the more pronounced degradation in mortar sections compared to their cement paste counterparts, despite the paste exhibiting higher total porosity (Table 2). This counterintuitive result can be attributed to the greater effective porosity in mortar due to the presence of interfacial transition zones (ITZs) between aggregates and the matrix. These ITZs, characterized by higher capillary porosity and larger pore throats (sometimes reaching up to 4 µm) facilitate the diffusion of sulfur species and allow microbial cells to infiltrate and colonize the material. In contrast, cement paste, despite its higher overall porosity, has a denser microstructure with smaller and less connected pores, limiting acid ingress, microbial penetration, and the formation of expansive sulfate phases. In addition, cement pastes contain a higher volume fraction of reactive hydrates per unit volume (due to the absence of aggregates), providing greater acid-buffering capacity. This larger reservoir of alkaline phases (portlandite, C-S-H, AFm) enhances neutralization of sulfuric acid and further delays the progression of degradation fronts compared to mortars. As supported by
literature findings [52, 53, 57], these combined morphological and chemical buffering features govern both physical diffusion and biodeterioration dynamics, especially during early degradation. The structural heterogeneity, particularly near aggregates, and increased microcrack susceptibility in mortars further exacerbate their vulnerability. These insights are essential for improving durability design in real-world sewerage environments.

**4.3. Size Effect and Its Implications for Degradation Kinetics**

The use of both 2x2x2 cm³ and 4x4x4 cm³ specimens provided critical insight into the influence of sample size on degradation kinetics. Smaller specimens deteriorated more rapidly, with CEM I showing complete disintegration at 180 days. The larger samples retained enough integrity for advanced analyses, but still followed the same zonation and degradation patterns.

The thickness of the degraded zones for both specimen sizes was estimated using an area-based approach (surface area divided by length), and the corresponding values are reported in Table 3.

These observations reflect a surface-to-volume ratio effect, where smaller specimens have the same intrinsic buffering capacity as larger ones, but the total amount of buffering material is proportionally lower, and diffusion paths for aggressive species are shorter. The higher exposure per unit volume accelerates chemical transformation. Similar findings have been reported by El-Hachem *et al.* [58], confirming that smaller prisms exhibit faster expansion and degradation onset compared to larger ones.

These results highlight the importance of scaling considerations in both laboratory testing and in interpreting accelerated durability studies.

**4.4. Quantification of Alteration Zones in Mortar and Cement Paste Sections**

To quantitatively assess the extent of biochemical degradation across the different cementitious materials, the total depth of the altered zone was measured on polished cross-sections using ImageJ software. For CEM I and CEM V, this comprised zones 1, 2, and 3 identified via SEM-EDS; for CAC, zones 1 and 2 were considered. The thickness was estimated using an area-based approach: the surface area of the altered region was quantified using ImageJ software and divided by its known longitudinal dimension, yielding an average width (i.e., thickness) of the degraded zone. The values reported in Table 3 are therefore geometrically derived from single cross-sections for each condition. Because this method does not rely on multiple independent measurements of the same parameter, statistical deviations (e.g., standard deviations) cannot be meaningfully calculated, which explains their absence in the table. These results nonetheless confirm that degradation is consistently more pronounced in mortars than in cement pastes, regardless of binder type or exposure duration.
At T0 (15 days of abiotic H₂S exposure), sulfur ingress was already evident in the outer layers of all specimens. This early diffusion was more pronounced in mortars than in cement pastes. For instance, CEM I mortar exhibited an altered depth of 2.34 mm, compared to 1.30 mm in its cement paste counterpart. Similar trends were observed for CEM V (2.42 mm in mortar vs. 1.29 mm in paste) and CAC (0.68 mm vs. 0.37 mm).

After 90 days of microbial exposure, degradation significantly intensified, particularly in Portland-based binders. The altered zone in CEM I mortar reached 2.91 mm, compared to 1.46 mm in the corresponding cement paste. CEM V followed a similar trend (2.68 mm in mortar vs. 1.96 mm in paste). CAC remained relatively stable, with altered depths of 1.31 mm in mortar and 0.54 mm in paste.

Due to the complete deterioration of the smaller 2x2x2 cm³ CEM I specimens at 180 days, larger 4x4x4 cm³ samples were used for final measurements. These showed a maximum altered depth of 6.65 mm for CEM I mortar, 5.43 mm for its paste; CEM V reached 5.43 mm (mortar) and 2.93 mm (paste). CAC again demonstrated the highest resistance, with 2.00 mm and 0.96 mm, respectively.

These trends align with macroscopic observations (Section 3.1), where CEM I showed early swelling and mass loss, while CAC remained dimensionally stable. Furthermore, the findings line up with long-term *in situ* investigations conducted on cylindrical mortar specimens (60 mm diameter) exposed for 49 months in sewer environments [31, 32], where altered depths were as follows:

* Low H₂S concentrations (1.3 ppm): 4.4 mm (CEM I), 4.8 mm (CEM V).

* Higher H₂S concentrations (14 ppm): 8.1 mm (CEM I), 9.9 mm (CEM V), 1.5 mm (CAC).

Notably, the accelerated test reproduced comparable degradation profiles and magnitudes within just 180 days, demonstrating the method's relevance and predictive capability for aggressive sewer conditions.

In this study, 2x2x2 cm³ mortar specimens were periodically analyzed to track degradation, while 4x4x4 cm³ specimens were assessed only at 180 days. The smaller specimens exhibited faster damage: CEM I failed completely, while altered depths for CEM V and CAC reached 3.58 mm and 1.86 mm, respectively. For the larger specimens, values were higher: 6.65 mm (CEM I), 5.43 mm (CEM V), and 2.00 mm (CAC). This confirms the influence of specimen size on degradation, as smaller samples, due to their higher surface-to-volume ratio and shorter diffusion paths, deteriorate more rapidly.
Similar trends were reported by El-Hachem *et al.* [58], who observed earlier sulfate-induced expansion in small prisms (1x1x10 and 2x2x16 cm³, around 130 days), compared to larger ones (4x4x16 cm³), where expansion began only after 400 days. This size effect was attributed to the fact that sulfate attack becomes significant only when a critical proportion of the cross-section is damaged by ettringite and/or gypsum formation, a threshold that is reached more rapidly in smaller volumes due to faster ingress of aggressive ions.

From a chemical standpoint, this behavior can be linked to the buffering capacity of the intact core. Larger specimens maintain their alkaline pore solution for longer due to slower OH⁻ leaching, thereby delaying the precipitation of expansive sulfate phases. In smaller specimens, rapid leaching accelerates ettringite and gypsum formation, promoting earlier and more severe degradation.

To facilitate microstructural analysis at 180 days, both small (2x2x2 cm³) and large (4x4x4 cm³) specimens were analyzed. As expected, the CEM I and CEM V formulations exhibited a marked size effect: the smaller the specimens the deeper the altered zones due to their higher surface-to-volume ratio, shorter diffusion paths, and reduced buffering capacity, which collectively accelerate sulfur ingress and the formation of secondary sulfate phases. The larger specimens retained a more substantial intact core, resulting in a smaller measured degraded thickness, although the mineralogical zonation remained identical. In contrast, CAC showed only minimal differences between the two specimen sizes, reflecting its intrinsically slower degradation kinetics and the overall limited progression of the alteration front in this binder.

<table><caption>Table 3: Depth of the chemically altered zone (in mm) measured in the cement paste and mortar regions of the specimens at different exposure durations. Values at T0 and 90 days were obtained from small samples (2x2x2 cm³), while values at 180 days were measured on both small and larger specimens (2x2x2 / 4x4x4 cm³).</caption><thead><tr><th></th><th></th><th>Cement Paste (mm)</th><th>Mortar (mm)</th></tr></thead><tbody><tr><td rowspan="3">T0</td><td>CEM I</td><td>1.30</td><td>2.34</td></tr><tr><td>CEM V</td><td>1.29</td><td>2.42</td></tr><tr><td>CAC</td><td>0.37</td><td>0.68</td></tr><tr><td rowspan="3">90 d</td><td>CEM I</td><td>1.46</td><td>2.91</td></tr><tr><td>CEM V</td><td>1.96</td><td>2.68</td></tr><tr><td>CAC</td><td>0.54</td><td>1.31</td></tr></tbody></table>
<table><tr><td rowspan="3">180 d<br>(2x2x2 /<br>4x4x4<br>cm³)</td><td>CEM I</td><td>- / 5.43</td><td>- / 6.65</td></tr><tr><td>CEM V</td><td>2.2 / 2.93</td><td>3.58 / 5.43</td></tr><tr><td>CAC</td><td>0.88 / 0.96</td><td>1.86 / 2.00</td></tr></table>

### 4.5. Comparison between accelerated and *in situ* exposure

To evaluate the representativity and predictive potential of the accelerated protocol, the degradation results obtained in this study were compared to those from previous in situ investigations by Ayoub *et al.* [31, 32], where 60 mm-diameter mortar specimens were exposed for 49 months in real sewer environments under low (1.3 ppm) and high (14 ppm) H₂S concentrations.

To ensure consistency, the altered thicknesses measured microscopically in all studies were normalized to a one-year exposure period. Acceleration factors were then calculated as the ratio between degradation rates observed in the accelerated test (250 ppm H₂S for 190 days) and those observed in the *in situ* studies. The results are summarized in Table 4:

<table><caption>Table 4: Altered thicknesses normalized to 1 year (mm/year), based on microscopic observations, in cementitious materials after accelerated testing (250 ppm H₂S, 190 days) compared with *in situ* exposures (49 months) under low (1.3 ppm) and severe (14 ppm) H₂S concentrations. Acceleration factors are calculated as the ratio of accelerated to *in situ* degradation.</caption><thead><tr><th>Cement Type</th><th>Normalized Altered thickness - Accelerated test</th><th>Normalized Altered Thickness - In Situ (14 ppm) [32]</th><th>Acceleration Factor (Accel./In Situ 14 ppm)</th><th>Normalized Altered Thickness - In Situ (1.3 ppm) [31]</th><th>Acceleration Factor (Accel./In Situ 1.3 ppm)</th></tr></thead><tbody><tr><td>CEM I</td><td>12.78 mm</td><td>1.98 mm</td><td><strong>6.5</strong></td><td>1.08 mm</td><td><strong>12</strong></td></tr><tr><td>CEM V</td><td>10.43 mm</td><td>2.42 mm</td><td><strong>4.5</strong></td><td>1.17 mm</td><td><strong>9</strong></td></tr><tr><td>CAC</td><td>3.84 mm</td><td>0.37 mm</td><td><strong>10.5</strong></td><td>-</td><td>-</td></tr></tbody></table>

From this comparison, several important conclusions can be drawn:

* The accelerated test produced comparable or even higher degradation levels in just 190 days than those observed after more than four years of *in situ* exposure, confirming its effectiveness in simulating long-term degradation under severe conditions.

* Based on normalized values, the estimated acceleration factor is approximately 5 for Portland cements under high H₂S concentrations and up to 10 under low H₂S
concentrations. For CAC, a factor of approximately 10.5 was observed under high H₂S exposure, although further long-term data are needed to confirm this value.

* Despite the intensified exposure conditions (250 ppm H₂S), the degradation patterns observed, particularly zonation and phase evolution, were consistent with those from long-term field exposure. This validates the test's ability to reproduce relevant degradation mechanisms within a compressed timeframe.

* It is important to note that the altered depths in both the accelerated and *in situ* studies were estimated from cross-sectional microscopy. These values likely underestimate total damage, as they do not account for material loss due to spalling or crumbling during exposure. The reported thicknesses should thus be interpreted as conservative estimates.

* Even under highly aggressive accelerated conditions, CAC exhibited limited degradation, consisted with its superior performance observed in field conditions.

* The higher acceleration factor calculated for CAC may reflect its initially low degradation rate under field conditions, combined with a slightly more sensitive response to aggressive laboratory conditions. While CAC remains the most resistant formulation overall, its dense microstructure and limited porosity may have delayed early degradation *in situ*, resulting in an apparently greater acceleration when exposed to concentrated H₂S under forced laboratory conditions. Further research is needed to verify this trend under varying durations and exposure regimes.

In summary, the results confirm that the accelerated test protocol provides a robust, predictive, and scalable tool for assessing the long-term durability of cementitious materials in sewer environments. Its ability to replicate mineralogical and structural degradation mechanisms within a significantly shorter timeframe makes it particularly useful for rapid screening and material selection in the context of wastewater infrastructure design.

**5. Conclusion**

This study presents a comprehensive multi-scale assessment of the biodeterioration of cementitious materials under accelerated exposure to a hydrogen sulfide (H₂S)-enriched microbial environment. Through the integration of macroscopic monitoring (surface pH, mass, and dimensional variations), SEM-EDS microstructural analysis, and mineralogical characterization via Raman spectroscopy coupled with chemometric techniques (PCA and MCR-ALS), the deterioration mechanisms of three cement types (CEM I, CEM V, and CAC) were evaluated in both mortar and cement paste configurations.
The findings confirm the extreme vulnerability of CEM I, which exhibited rapid surface disintegration, complete calcium depletion, and widespread formation of sulfate-bearing phases such as gypsum and ettringite. CEM V, a blended cement, showed improved resistance, with degradation confined to the surface and preservation of the inner matrix. In contrast, CAC demonstrated the highest resistance to biogenic sulfuric acid attack, with minimal elemental migration and limited sulfate-induced transformation, even after 180 days of exposure.

One of the most significant findings is the identification of progressive degradation zonation across all specimens, particularly in Portland-based formulations (CEM I and CEM V). The transition from surface-enriched sulfate zones to an unaltered core was consistently observed using both SEM-EDS and Raman imaging. These zonations were notably more developed in mortar than in cement paste, despite the latter's higher total porosity. This behavior was attributed to greater pore connectivity, the presence of interfacial transition zones (ITZs), and increased structural heterogeneity in mortars, all of which facilitate the diffusion of aggressive species and accelerate degradation.

Importantly, micro-Raman spectroscopy proved to be a crucial tool in this investigation, offering high spatial resolution and chemical specificity for the identification of both pristine hydration products and newly formed degradation phases. Combined with chemometric processing, this technique enabled the clear visualization of sulfate-related mineralogical transformations and the progression of the biodeterioration front, which are not achievable through macroscopic analysis alone. Its application thus significantly enhanced the depth and precision of the degradation assessment.

Moreover, the study showed that the degradation trends identified in smaller specimens were reproducible in larger ones, validating the scalability and robustness of the accelerated testing protocol. When compared to previous long-term *in situ* field studies over 49 months [31, 32], the accelerated test reproduced similar degradation depths and mineralogical signatures in just 180 days. Based on normalized degradation rates, the estimated acceleration factor of the test ranged from approximately 5 to 10 for Portland cements, and reached 10.5 for CAC, depending on the H₂S exposure level (1.3 ppm vs. 14 ppm). These values reinforce the predictive value of the protocol for simulating extended field exposure within a significantly reduced timeframe.

In summary, this work underscores the importance of binder composition, microstructure, and specimen scale in governing resistance to microbial sulfate attack. The proposed accelerated protocol offers a reliable, efficient, and representative approach for screening and optimizing cementitious materials intended for aggressive sewer environments. These insights can inform material selection, performance-based design, and durability forecasting in wastewater
infrastructure applications, supporting the development of more resilient and sustainable construction solutions.

## Data Availability

Data will be provided upon request

## Appendix

<table><caption>Table A-1: Statistical summary of H₂S concentration (ppm) during the six-month exposure period.</caption><thead><tr><th></th><th>Mean</th><th>Median</th><th>1<sup>st</sup> quartile</th><th>3<sup>rd</sup> quartile</th><th>Min</th><th>Max</th></tr></thead><tbody><tr><td>H<sub>2</sub>S (ppm)</td><td>250</td><td>337</td><td>98</td><td>365</td><td>0</td><td>1323</td></tr></tbody></table>

<figure><img src="image_37.png" /><figcaption>Figure A-1: Overview of a CEM V specimen after 180 days of exposure, showing the regions selected for Raman mapping. Mapped areas ranged from approximately 3.5 x 5.5 mm to larger regions depending on the extent of degradation. Red rectangles indicate the selected sections, which are shown at higher magnification (10× objective) for detailed investigation.</figcaption></figure>

## Acknowledgments

"This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie COFUND grant agreement No 101034248." The authors gratefully acknowledge Béatrice Desrues for her invaluable assistance in conducting the SEM-EDS acquisition and SIAAP for its technical support in providing the activated sludge.

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Declaration of interests

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: