Biodeterioration Study of Cementitious Materials During Sewage Treatment Processes

Abstract

The relationship between microbial communities and mineralogical/mechanical changes was studied regarding the biodeterioration of Portland cement (PC) and calcium sulfoaluminate cement (CSAC) in a wastewater treatment plant. An in situ experiment was conducted by submerging 12 independent PC and CSAC specimens in a sand-trap structure for 10, 30, 75, 150, and 240 days. The microbiological analyses of the 16S rRNA genes of bacteria and Archaea from the biofilms and the geochemical analysis were performed on the studied specimens. The results showed that while there were characteristic changes in PC specimens over time, CSAC specimens showed few biodeterioration effects. The dominant bacteria identified from the biofilms of specimens belonged to the classes of Gammaproteobacteria (8.4–32.4%), Bacilli (1.6–21.6%), Clostridia (4–15.4%), Bacteroidia (2–18.8%), Desulfovibronia (0.5–19%), Campylobacteria (0.4–26.8%), and Actinobacteria (1.8–12.8%). The overall relative abundance of the bacteria linked to biodeterioration processes increased to more than 50% of the total bacterial communities after 75 days of sewage exposure and was found to be strongly correlated with several PC deterioration parameters (e.g., mass loss, calcite and ettringite minerals), whereas no significant correlation was revealed between these genera and CSAC characteristics.

1. Introduction

Cement-based materials have long been used in constructing wastewater treatment plant (WWTP) structures due to their properties of excellent watertightness, durability, and mechanical strength. Concerns have existed for decades about the biodeterioration of wastewater infrastructures due to microbial-induced corrosion (MIC). Nevertheless, there is still little consensus on the methods of design and material specifications of WWTP structures in terms of existing in such harsh environments and enduring long-term service [1,2]. This problem has been reported primarily in sewer pipes and several units of WWTP facilities, such as wastewater tanks and pre-treatment units [3]. In wastewater-related infrastructures, including WWTPs, sulfide oxidation by Thiobacillus spp. is described as the major cause of the biodeterioration of Portland cement due to the corrosive nature of produced H₂SO₄ [4]. However, due to the abundant nature of microbial communities in domestic sewage [5], other bacterial communities that are less mentioned in the literature can also play an important role in cement deterioration. For example, certain bacteria that are used in the biological treatment of wastewater in removing sulphate, organic matter, and nitrate [6,7] may also contribute to the biodeterioration process by producing different metabolic products (e.g., H₂S and H₂SO₄), which react with cement minerals. Moreover, bacteria that are involved in fermentation can result in the generation of organic acids (e.g., acetic acid) and CO₂, which can also influence concrete biodeterioration [8].

In Portland cement (PC)-based infrastructures, biodeterioration processes are caused by the reactions of microbial metabolic products (e.g., H₂SO₄, H₂CO₃ or organic acids) and alkaline mineral components, e.g., portlandite (Ca(OH)₂ and calcium silicate hydrate (C-S-H), resulting in the formation of secondary minerals i.e., gypsum (CaSO₄·2H₂O), ettringite (Ca₆Al₂(SO₄)₃(OH)₁₂·26H₂O), and thaumasite (Ca₃Si(OH)₆CO₃SO₄·12H₂O) [9,10,11]. The consequences of concrete biodeterioration include pH reduction, crack formation, mass loss, and the loss of engineering properties. According to the literature [11,12], the reduction in pH makes the concrete surface more susceptible to the colonization of acidophilic bacteria communities, whereas the formation of secondary minerals (e.g., gypsum and ettringite) causes volume expansion, leading to physical disintegration and cracks [13,14].

Alternative cement materials to overcome the drawbacks of Portland cement in the biodeterioration process in WWTPs are still being tested. One of them is calcium sulfoaluminate cement (CSAC) [15]. This cement contains a mineralogical composition that is stable and not so reactive against external compounds such as metabolic products from microorganisms. Its mineralogical composition contains ye’elimite (Ca₄Al₆O₁₂SO₄), belite (Ca₂SiO₄), and anhydrite (CaSO₄) as clinker (also known as unreacted) minerals as well as ettringite and gibbsite as the main hydrated minerals [16]. The absence of Ca(OH)₂ and C-S-H (Ca₅Si₆(OH)₂·4H₂O) makes the CSAC concrete more resistant to attacks from H₂SO₄ and H₂CO₃ generated by microorganisms. Because of this, various studies, e.g., [17,18], suggest that CSAC-based materials perform better in biodeteriorating conditions due to their excellent biogenic resistance properties. However, CSAC is not yet commonly used in the construction of WWTP structures due to its high costs caused by the limited availability and economics of its aluminate source, i.e., ye’elimite (Ca₄(AlO₂)₆SO₄), which is its principal constituent, comprising of (30–70%) of the mineralogical composition [15].

The present study aimed to (1): analyze the maturation of the biofilms developing on PC and CSAC specimens, (2): identify the deterioration effects in PC and CSAC, and (3): reveal the relationship between the microbial communities and the primary physical and chemical cement characteristics. This study employed a combined approach of molecular biology techniques, geochemical analysis, and engineering tests to understand the relationship between microorganisms and cement characteristics, including their mineralogical and mechanical properties during cement biodeterioration in WWTPs.

2. Materials and Methods

2.1. Cement Specimens’ Preparations

Two cement types were used, i.e., Portland cement (PC) of grade 52.5 N and calcium sulfoaluminate cement (CSAC). PC powder comprised 95% Portland clinker and 5% gypsum, whereas CSAC powder comprised 80% CSA clinker and 20% anhydrite. Twelve cement paste specimens comprising 6 PC and 6 CSAC specimens were prepared, and one specimen from each cement type was kept in the laboratory as a reference. The cement specimens were prepared in a Le Chatelier-ring mold by mixing 500 g of cement powder with 180 mL of deionized water according to the European test standard EN 196-3:2016 [19] for PC and the European Assessment Document (EAD 150001-00-0301) 2017 for CSA [20]. The water-to-cement ratio for the specimens was 0.36. Their dimensions were 30 mm in diameter by 30 mm in height, and they were cylindrical in shape. The chemical composition of the used cement types and the reference specimens’ initial mineralogical composition are shown in

Table 1. Chemical composition (mass, m/m%) of the used cement: PC and CSAC.

Materials (m/m%)	SiO2	Al2O3	Fe2O3	CaO	MgO	K2O	Na2O	SO3	TiO2	Cl−	LOI*	Total
PC	18.75	5.59	3.53	64.17	2.34	0.25	0.02	3.35	0.39	0.02	1.23	99.64
CSAC	6.18	23.49	1.28	36.64	4.80	0.36	0.75	21.54	0.36	0.10	0.70	96.20

LOI*: Loss on ignition of the initial cement materials.

and

Table 2. Initial mechanical characteristics and mineralogical compositions in the two reference specimens of PC and CSAC. nd—not detected and ne—not expected, respectively.

Property	Cement Properties	PC Specimen	CSAC Specimen
Mechanical Properties	Surface pH	8.95	9.68
	Compressive Strength	68.9 MPa	115.7 MPa
Mineralogical Compositions
Secondary Minerals	hydrocalumite (Ca2Al(OH)6Cl·2H2O)	2%	ne
	calcite (CaCO3)	4%	nd
	monosulfoaluminate (Ca4Al2(SO4)(OH)12·6H2O)	nd	ne
	ettringite (Ca6Al2(SO4)3(OH)12·26H2O)	17%	nd
Hydrated Minerals	katoite (Ca3Al2O6·6H2O)	10%	ne
	CASH (Ca12Al2Si18O51(OH)2·18H2O)	2%	ne
	C-S-H (Ca5Si6O16(OH)2·4H2O)	11%	ne
	portlandite (Ca(OH)2)	28%	nd
	ettringite (Ca6Al2(SO4)3(OH)12·26H2O)	nd	58%
	gibbsite (Al(OH)3)	nd	6%
Clinker Minerals	Periclase (MgO)	nd	3%
	merwinite (Ca3Mg(SiO4)2)	4%	10%
	brownmillerite (Ca4Al2Fe2O10)	1%	ne
	akermanite (Ca2Mg(Si2O7))	1%	nd
	ye’elimite (Ca4Al6O12SO4)	ne	7%
	anhydrite (CaSO4)	nd	7%
	belite (Ca2SiO4)	3%	9%
	alite (Ca3SiO5)	17%	ne

, respectively. The prepared specimens were cured for 28 days at a temperature of 20.0 ± 1.0 °C and relative humidity of ≥90% in the laboratory to achieve the required standard conditions for their preparation.

2.2. Experimental Design

The specimens were submerged into sewage in the sand trap of the Velence WWTP near Velence Lake in Hungary from June 2023 to February 2024 for an in situ experiment. Two-meter-long PVC pipes 150 mm in diameter were used as specimen holders and fixed at the top to prevent them from being flushed away by sewage (Figure 1). Different exposure times were used, i.e., 10, 30, 75, 150, and 240 days, from 16 June 2023 to 7 February 2024. Afterwards, cement specimens were removed for microbiological and geochemical analyses. The specimen codes and their names in brackets are defined as follows: PC specimens: R (reference PC), PC1 (PC after 10 days), PC2 (PC after 30 days), PC3 (PC after 75 days), PC5 (PC after 240 days); CSAC specimens: CR (reference CSAC), CSAC1 (CSAC after 10 days), CSAC2 (CSAC after 30 days), CSAC3 (CSAC after 75 days), CSAC4 (after 150 days), and CSAC5 (CSAC after 240 days). The PC4 (PC after 150 days) is missing because it was flushed away by a high sewage flow during the exposure experiment.

2.3. Sample Preparation for Analysis

After each intended period of sewage exposure, two specimens (i.e., 1 PC and 1 CSAC specimen) were removed from the sample holder and carefully packed into a sterile container to avoid contamination. In the laboratory, biofilms were carefully removed from each specimen with a sterile toothbrush and washed in a 20 mL Ringer solution containing salts of NaCl, CaCl₂·2H₂O, KCl, and pH buffer (NaOH). The biofilms were then stored at −20 °C until analysis. After the removal of the biofilm, specimens were autoclaved to avoid contamination of the analytical instruments during geochemical analyses. Then, they were sealed in three layers of light-density polyethene (LDPE) plastic bags and air-dried in a desiccator containing silica gel for a week until geochemical analysis. The original wastewater was also sampled from the study site with the following compositions in mg/L: biological oxygen demand (BOD₅) (452), chemical oxygen demand (COD) (911), Kjeldahl nitrogen (85.40), nitrite (NO₂-N) (0.072), nitrate (NO₃-N) (0.47), total phosphorus (12.61), pH (7.89), total suspended solids (TSS) (444.88), and total nitrogen (85.68).

2.4. Microbiological Analysis

2.4.1. Biofilm Sampling, DNA Extraction, PCR Reaction, and Amplicon Sequencing

The DNA extraction was performed using the DNeasy ^®PowerSoil^® Pro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The samples were thawed and filtered using a cellulose 0.22 µm MCF membrane filter. Mechanical cell disruption was carried out by a Powerlyzer 24 homogenizer (Qiagen, Germany) operated at 30 Hz for 2 min. Polymerase chain reactions (PCRs) of the isolated DNA were performed in a 20 μL final volume containing the following components: 1 µL of DNA template, 4 µL HB Buffer, 4 µL dNTP nucleotides, 0.2 µL forward (341 F) and reverse primers (805 R), 0.4 µL BSA reaction buffer, 0.2 µL Phusion polymerase enzyme, and 10 µL dH₂O. The V3–V4 region of the 16S rRNA gene was amplified using the following primers: Bact-341F (5′-CCT ACG GGN GGC WGC AG-3′) [21] and 805R (5′-GACTACHVGGGTATCTAATCC-3′) for Bacteria and Arch-519F (5′-CAGCMGCCGCGGTAA-3′) and Arch-855R (5′-TCCCCCGCCAATTCCTTTAA-3′) for Archaea [22]. All the PCR reactions were performed by the following thermocycling program: 5 min of initial denaturation at 98 °C, followed by 25 amplification cycles of 40 s at 95 °C, 30 s at 55 °C for Bacteria and 60 °C in the case of Archaea, and 1 min at 72 °C and extension at 72 °C for 10 min. The quality and quantity of PCR products were checked by agarose gel electrophoresis and a QubitTM Fluorometer (dsDNA HS Assay kit). Only samples that showed a positive PCR reaction producing ≥ 5 ng/µL DNA were sent for Next-Generation sequencing. Amplicon sequencing was performed at SEQme Ltd. (Prague, Czech Republic) using the Illumina Novaseq 6000 platform-Shareseq package (Illumina, San Diego, CA, USA) in a 2 × 250 bp paired-end format.

2.4.2. Bioinformatic Analysis

The bioinformatic analyses of the raw sequences were performed using Mothur software version v.1.48.1 [23], and the Miseq SOP pipeline was followed (https://mothur.org/wiki/miseq_sop/, accessed on 16 February 2024). FASTQ forward and reverse sequence reads were imported into the Mothur software, combined, and aligned using the Silva database (silva.nr_v138_1.align) [24], whereas the taxonomic affiliation was made by the ARB-SILVA SSU NR reference database (silva.nr_v138_1.tax) [25] at the cut-off of 80. The operational taxonomical units (OTUs) were assigned at 97% similarity threshold levels [26], whereas the alpha diversity was calculated as published by Toumi et al. [22]. The sequence reads were deposited in the NCBI SRA database and are accessible through BioProject accession number PRJNA1208061. The relative abundances of different taxa were then calculated in Excel for further statistical analyses.

2.5. Geochemical Analyses

2.5.1. Specimens Morphology and Microstructure

Scanning electron microscopy (Hitachi TM 4000 plus model, Shanghai, China) was used to examine the morphology and microstructure of specimens after sewage exposure. For morphological analyses, unpolished surfaces coated with gold were analyzed by using a secondary electron detector (SEM-SE), whereas, in microstructural analysis, the polished surfaces coated with carbon were analyzed using a backscattered electron detector (SEM-BSE), both operated at 15 kV/mode 3 with a low volume due to the porous nature of cement specimens. During the SEM analysis, small sections were used. They were cut from the original sample using a diamond saw and coated using the SPI-Module sputtering coating method to a thickness of 12 nm for carbon and 5 nm for gold.

2.5.2. Specimen Mineralogy

The mineralogical composition of the studied specimens was analyzed using an X-ray diffraction machine (Bruker D2 phaser XRD powder diffractometer model, Billerica, MA, USA). The samples were prepared by manually grinding specimens into powder (<63 µm) using a mortar and pestle. Then, mineral phases were identified and quantified using the search/match and Rietveld refinement procedures explained in our previous publication [14].

2.6. Compressive Strength of the Specimens

The compressive strength of the specimens was measured by a calibrated press (Toni Technik Baustoffprüfsysteme GmbH, Berlin, Germany) at the CEMKUT material lab (Budapest, Hungary). The mass and geometrical dimensions were measured using a calibrated digital scale and caliper. The measurement was performed according to the European Standard EN 196-1:2016 [19]. After the test, the strength values of each specimen were calculated by dividing the maximum load at failure by the cross-sectional area.

2.7. Statistical Analysis

Principal component analysis (PCA) was performed using Statigraphics.v18 to reveal the relationship between microbial communities and mineralogical changes in cement over time. The PCA axes with minimum eigenvalues ≥ 1 were analyzed. Pearson correlation and nonmetric-multidimensional scaling ordination (NMDS) analyses were performed using Stratigraphics.v.18, and PAST.v4.17 software was used to confirm the PCA results. The minerals used in the statistical analyses were selected from three main categories: clinkers, hydrated, and secondary minerals, as shown in Table 2. The individual bacterial genera were also selected from three groups, i.e., sulfate-reducing bacteria (SRB), sulfide-oxidizing bacteria (SOB), and fermentative bacteria, based on their abundances. All data were normalized to values between 1 and 0 before the statistical analyses.

3. Results

3.1. Microbiological Results

3.1.1. Bacterial Community Structure

Figure 2a,b show the bacterial diversity and community structure at the phylum level of the wastewater sample and the specimen biofilm samples studied. The bacterial diversity in both cement-type specimens increased with the maturation of biofilms, except for specimens after 240 days of sewage exposure.

The bacterial community structure of the wastewater sample was dominated by Actinobacteriota (33.47%) and Chloroflexi (27.27%), followed by Firmicutes (16.95%), Proteobacteria (8.67%), Planctomycetota (2.6%), Gemmatimonadota (1.49%), and Synergistota (1.02%), whereas in the cement specimens, the bacterial community structure changed with the increase in time. The ratio of different phyla in the biofilm after 10 days of sewage exposure changed, whereby the dominant phyla were Proteobacteria (31.23% for PC and 27.44% for CSAC), followed by Firmicutes (30.47% for PC and 26.02% for CSAC), respectively. For specimens at 10 days of exposure, the ratios of some bacterial phyla (e.g., Proteobacteria, Firmicutes, and Desulfobacterota) increased, whereas others (e.g., Actinobacteriota and Chloroflexi) decreased as compared to the wastewater sample. After 30 days, in the case of PC specimens, the Firmicutes remained dominant but slightly decreased to 23.74%, whereas Bacteroidota was the second most dominant and increased along with Desulfobacterota and Synergistota. The relative abundances of other phyla (e.g., Proteobacteria and Actinobacteriota) decreased. In the case of CSA specimens after 30 days (in CSAC2), Firmicutes and Actinobacteriota were dominant and increased together with Desulfobacterota and Synergistota, whereas Proteobacteria decreased significantly. After 75 days, in the case of PC specimens (in PC3), the bacterial community structure was dominated by Desulfobacterota and increased to 23.54%, together with Synergistota. In contrast, Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteriodota decreased altogether. In the case of CSAC specimens at 75 days of exposure (in CSAC3), Firmicutes and Bacteroidota remained dominant, but the former decreased with Proteobacteria and Actinobacteriodota, while the latter increased with Desulfobacterota and Synergistota. After 240 days of sewage exposure (i.e., PC5 and CSAC5), the bacterial community structure of both PC and CSAC specimens was dominated by Proteobacteria, Firmicutes, and Campylobacterota. In contrast, the relative abundances of Actinobacteriota, Bacteroidota, and Synergistota decreased.

Among the Proteobacteria, the ratio of Gammaproteobacteria increased between the initial 10 days and after 240 days of sewage exposure in both cement types, whereas Alphaproteobacteria decreased. Among the Firmicutes, the ratio of Bacilli increased between the initial 10 days and 240 days, whereas Clostridia decreased in both specimens. Moreover, the ratios of Desulfovibronia, Bacteroidia, and Actinobacteria decreased between 10 days and 240 days of sewage exposure, whereas the Campylobacteria increased, as shown in Figure 2. When compared at 240 days, both PC and CSAC specimens had the highest abundances of Gammaproteobacteria, Bacilli, and Campylobacteria. However, PC specimens showed a higher abundance of Gammaproteobacteria (32.38%) than CSAC specimens.

Several bacterial genera which are presumably associated with cement biodeterioration processes were revealed in the present study. These include sulfate-reducing bacteria (SRB), sulfide-oxidizing bacteria (SOB), and fermentative bacteria, as detailed in

Table 3. Heatmap showing the relative abundances of the most abundant genera of the sulfate-reducing (SRB), sulfur/sulfide-oxidizing (SOB), and fermentative bacteria in the biofilms of Portland cement (PC) and calcium sulfoaluminate cement (CSAC) specimens.

Groups	Genera				Class					WW	PC Specimens				CSAC Specimens
											PC1	PC2	PC3	PC5	CSAC1	CSAC2	CSAC3	CSAC4	CSAC5
Sulfate reducing bacteria (SRB)	Desulfomicrobium				Desulfovibronia					0.12	2.09	6.68	18.30	0.32	2.48	8.16	11.50	6.40	0.30
	Desulfobulbus				Desulfobulbia					0.08	1.57	2.23	3.20	0.11	0.97	1.64	2.65	3.32	1.32
	Desulfovibrio				Desulfovibronia					0.17	1.03	0.36	0.37	0.12	0.92	0.59	0.30	0.17	0.03
	Desulfomonile				Desulfomonilia					0.00	0.18	0.10	0.37	0.03	0.09	0.14	0.48	0.60	0.23
	Sulfurospirillum				Campylobacteria					0.06	0.22	0.12	0.00	0.30	0.36	0.12	0.12	0.08	0.23
	Fusibacter				Clostridia					0.09	0.14	0.56	0.24	0.19	0.30	0.93	0.29	0.08	0.04
Sulfur oxidizing bacteria (SOB)	Bosea				Alphaproteobacteria					0.01	0.00	0.01	0.04	0.00	0.00	0.00	0.02	0.00	0.00
	Sulfuricurvum				Campylobacteria					0.00	0.00	0.00	0.02	0.00	0.02	0.00	0.00	0.00	0.00
	Sulfurimonas				Campylobacteria					0.05	0.00	0.26	0.00	0.12	0.00	0.07	0.00	0.07	0.27
	Thiothrix				Gammaproteobacteria					0.01	0.07	0.04	0.00	0.05	0.00	0.05	0.02	0.17	0.05
Fermenters	Trichococcus				Bacilli					1.42	18.13	5.77	1.12	19.84	11.15	6.16	0.68	4.48	25.01
	Macellibacteroides				Bacteroidia					0.08	5.42	12.07	10.51	1.35	7.72	1.06	14.05	5.98	1.68
	Acetobacterium				Clostridia					0.04	1.68	1.41	2.26	0.89	2.44	2.18	2.49	2.98	1.01
	Christensenella				Clostridia					0.21	1.05	3.95	4.99	0.12	1.49	2.70	6.39	4.76	1.30
	Clostridium_sensu_stricto				Clostridia					0.26	1.40	1.37	2.54	0.26	1.75	2.18	2.79	2.32	1.24
	Butyricicoccus				Clostridia					0.00	0.00	0.00	0.00	0.07	0.19	0.03	0.00	0.00	0.03
	Ruminococcus				Clostridia					0.04	0.60	0.51	0.16	0.48	0.97	0.79	0.34	0.68	0.41
	Propionivibrio				Gammaproteobacteria					0.00	0.30	0.81	0.65	0.49	0.44	0.16	0.58	0.95	0.19
	Enterococcus				Bacilli					0.35	0.56	0.14	0.07	0.87	0.34	0.00	0.05	0.12	0.00
	Streptococcus				Bacilli					0.00	0.30	0.19	0.00	0.04	0.28	0.05	0.02	0.58	0.04
	Mycobacterium				Actinobacteria					8.75	3.09	3.96	2.37	0.22	3.77	3.86	2.17	1.57	0.20
	Lactivibrio				Synergistia					0.13	0.16	0.45	1.34	0.07	0.13	1.69	1.80	1.80	0.66
	Romboutsia				Clostridia					1.67	0.41	0.37	0.73	0.11	0.17	1.18	1.57	1.08	0.20
	Peptococcus				Clostridia					0.00	0.03	0.09	0.14	0.03	0.05	0.30	0.34	0.27	0.03
	Intestinimonas				Clostridia					0.03	0.00	0.04	0.00	0.03	0.00	0.00	0.02	0.00	0.00
	Oscillospirales_ge				Clostridia					0.05	0.18	1.01	1.05	0.05	0.33	0.41	1.24	0.80	0.15
	Candidatus_Soleaferrea				Clostridia					0.00	0.03	0.10	0.06	0.00	0.00	0.00	0.11	0.08	0.00
	Faecalibacterium				Clostridia					0.76	0.58	0.41	0.04	0.38	0.51	0.05	0.01	0.07	0.22
	total relative abundances (SRB +SOB+fermenters)									14.38	39.21	43.02	50.57	26.55	36.85	34.51	50.04	39.40	34.85
	Others									85.62	60.79	56.98	49.43	73.45	63.15	65.49	49.96	60.60	65.15
	pH										8.93	8.62	8.5	7.75	8.84	9.3	8.3	8.09	8.84
	>50%	20–49%	10–19%	1–9%		0.1–1%	0–0.5%
Legend

. The most abundant genera from each of these groups were found to be SRBs (Desulfomicrobium spp. and Desulfobulbus spp.), SOBs (Thiothrix spp., Bosea spp. and Sulfurimonas spp.), and fermentative bacteria (Trichococcus spp., Acetobacterium spp., Macellibacteroides spp., Mycobacterium spp.). The total abundance of these bacterial groups (i.e., SRB, SOB, and fermentative bacteria) in the wastewater sample was 14.38%. It increased in PC specimens to 39.22%, 50.57%, and 27.60% after 10, 75, and 240 days of sewage exposure, whereas in CSAC specimens, it increased to 36.85%, 50%, and 34.85% after 10, 75, and 240 days of sewage exposure.

3.1.2. Archaeal Community Structure

The archaeal diversity index increased in the first 75 days and decreased after 240 days of exposure in both cement-type specimens, as shown in Figure 3. However, the PC specimens had higher archaeal diversity than CSAC specimens, except for the last specimen after 240 days (Figure 3).

In the wastewater sample, no archaeal community is reported in this study because the PCR products were insufficient for amplicon sequencing. However, in the biofilms of the cement specimens, the results showed that the dominant Archaea belonged to the class of Methanosarcinia, making up 87.73% and 92.49% of the entire archaeal community structure in PC and CSAC, respectively. Other dominant classes were Methanobacteria and Methanomicrobia, as shown in Figure 3. In Methanosarcinia, the dominant Archaea were Methanosaeta spp., Methanomethylvorans spp., and Methanosarcina spp. In PC, the relative abundance of Methanosarcinia increased with the maturation of biofilms from 34.10% in the sample exposed for 10 days to 87.73% after 240 days, whereas in CSAC, it increased from 39.87% in 10 days to 92.5% after 240 days. The relative abundance of Methanomicrobia also increased with the maturation of biofilms, except in the last sample after 240 days. In contrast, Methanobacteria decreased over time from 42.7% (after 10 days) to 9.3% (after 240 days) in PC and from 38% (after 10 days) to 3.3% (after 240 days) in CSAC specimens.

3.2. Geochemical Results

3.2.1. Mineralogical and Mechanical Properties

The variation in the mineralogical compositions of the studied cement specimens is presented in Figure 4. In PC specimens, there was a significant change in their mineralogical composition over time, whereby the amounts of some minerals were increasing, e.g., portlandite (Ca(OH)₂) and calcium silicate hydrate (C-S-H), whereas others, such as alite (Ca₃SiO₅) and belite (Ca₂SiO₄), were decreasing. Moreover, the varying amounts of secondary minerals such as ettringite (Ca₆Al₂(SO₄)₃(OH)₁₂·26H₂O), calcite (CaCO₃), and monosulfoaluminate (Ca₄Al₂(SO₄)(OH)₁₂∙6H₂O) were observed in PC specimens (Figure 4a). In contrast, the CSAC specimens showed few mineralogical changes over time (Figure 4b). Their mineralogical composition was dominated mainly by ettringite and gibbsite (Al(OH))₃, making up 65% of the total. The initial minerals (i.e., clinkers) of the CSAC specimens showed few changes in their amounts as compared to PC specimens, e.g., ye’elimite (Ca₄(AlO₂)₆SO₄) from 7% to 5%, belite (Ca₂SiO₄) from 9% to 8%, and anhydrite (CaSO₄) from 7% to 6% (Figure 4b). The only secondary mineral found in CSAC specimens was calcite (CaCO₃), which was observed in the last three specimens, i.e., CSAC3 (after 75 days), CSAC4 (after 150 days), and CSAC5 (after 240 days) (Figure 4b).

Regarding mechanical properties, the PC specimens showed an overall decrease in their compressive strength over time, with the last specimen after 240 days showing the lowest strength of 50.2 MPa (Figure 4a). In contrast, the CSAC specimens showed an irregular pattern of compressive strength values, with some decreasing and others increasing at different periods of exposure, e.g., 111.7–67.5 MPa between the reference and 30 days, 67.5–97.3 MPa between 30 and 75 days, 97.3–77.7 MPa between 75 and 150 days, and 77.7–83.7 MPa between 150 and 240 days (Figure 4b). However, the final strength value was 83.72 MPa, higher than the PC (i.e., 50.2 MPa).

3.2.2. Mass Loss and pH Reduction

Regarding the mass of the cement specimens shown (see Figure 2 and Figure 3,

Table 4. Mass loss (in g) and surface pH of the studied PC and CSAC specimens.

	Samples	Mass Loss (g)	%	pH
PC Specimens	Reference PC	–	–	8.95
	PC1 (10 days)	0.3	0.72	8.93
	PC2 (30 days)	0.5	1.2	8.62
	PC3 (75 days)	1.1	2.63	8.5
	PC5 (240 days)	0.1	0.24	7.75
CSAC Specimens	Reference CSAC	–	–	9.68
	CSAC1 (10 days)	0.2	0.48	9.3
	CSAC2 (30 days)	0.1	0.24	8.84
	CSAC3 (75 days)	1.2	2.86	8.3
	CSAC4 (150 days)	1.0	2.38	8.09
	CSAC5 (240 days)	0.5	1.19	8.84

), the mass loss gradually increased in specimens of both cement types from 10 to 75 days and decreased from 150 to 240 days. In both cases, the last specimens, after 240 days of sewage exposure, showed lower mass losses, i.e., 0.1 g (0.24%) in the case of PC and 0.5 g (1.19%) in the case of CSAC. Also, PC specimens’ surface pH values constantly decreased during exposure from 8.95 for the reference PC to 7.75 (after 240 days) for the last specimen (PC5), whereas in CSAC, the pH decreased from 9.68 for the reference to 8.84 for the last specimen (CSAC5) after 240 days of sewage exposure (Table 4).

3.2.3. Specimens Microstructure and Morphology

Figure 5 shows the SEM results of the microstructure and morphology of the reference and exposed cement specimens after 10 days, 75 days, and 240 days of sewage exposure. In the case of PC specimens, their microstructure (see first row) showed a decrease in white-color phases (i.e., clinker minerals) over time with sewage exposure. In contrast, the grey-color phases (i.e., hydrated minerals) increased over time. Moreover, their surface morphology (see second row) showed that the needle-shaped, hexagonal, and plate-shaped crystals were more visible with increasing sewage exposure.

In the case of CSAC specimens, the microstructure (see third row) looked alike, especially between the reference and after 10 days. However, there were visible micro-cracks after 75 and 240 days of sewage exposure. Regarding their morphology (see fourth row), numerous needle-like crystals were visible and identified as ettringite. Also, other crystals with spongy-like shapes were visibly observed during the 10, 75, and 240 days of exposure.

3.2.4. Relationship Between Microbial Communities and Cement Properties

In the case of the CSAC specimens, no characteristic deterioration was observed due to their stable mineralogical composition compared to the PC specimens, as shown in Figure 4. As a result, no significant relationship was revealed (i.e., p ≤ 0.05) between minerals and microbial communities, except in pH and Propionivibrio spp. (p = 0.03, R² = −0.91), as well as mass loss (p = 0.03, R² = −0.914).

In the case of PC specimens, the principal component analysis (PCA) showed that the PC specimens exposed for shorter periods, i.e., 10 days (PC1) and 30 days (PC2), were grouped closer to each other. In contrast, the ones exposed for longer periods, i.e., 75 days (PC3) and 240 days (PC5), were separated far from each other (Figure 6). Moreover, nonmetric multidimensional scaling ordinations based on Bray–Curtis dissimilarity distance (Figure 7) showed a clear separation between PC cement specimens at different exposure times and the relationships between several microbial communities and cement characteristics (e.g., mineral compositions and mass loss). In the group of secondary minerals, only calcite showed a significant correlation with Acetobacterium spp.(p = 0.02, R² = 0.98) and Desulfobulbus spp.(p = 0.05, R² = 0.95), whereas both clinker minerals (i.e., alite and belite) and hydrated minerals (i.e., C-S-H) showed no significant correlations (i.e., p > 0.05) with microbial communities, regardless of their strong positive or negative correlation values (Figure 8). However, further observation of the PCA and NMDS (Figure 6 and Figure 7) showed a close relationship between the mass loss of the PC specimens, calcite, Acetobacterium spp., and SRBs. Based on the correlation results (Figure 8), the mass loss significantly correlated with Sulfurospillilum spp. (p = 0.021, R² = −0.98), Desulfobacter spp. (p = 0.015, R² = 0.9849), and Desulfomicrobium spp.(p = 0.0051, R² = 0.99), whereas no significant correlation was observed between mass loss and cement minerals (e.g., C-S-H). Moreover, Thiothrix spp., Sulfurispirillum spp., and Trichococcus spp. were associated with ettringite and C-S-H minerals.

4. Discussion

In the present study, diverse bacterial communities that were identified from the biofilms of PC and CSAC specimens were involved in sewage treatment processes, such as sulfate reduction by sulfate-reducing bacteria (SRB), sulfide oxidation by sulfur-oxidizing bacteria (SOB), and anaerobic degradation of organic matter by fermenting bacteria, as detailed in Table 4. The metabolic products produced by these bacteria during sewage treatment, including H₂S, H₂SO₄, CO₂, and organic acids, can react with cement minerals, altering their compositions and leading to biodeterioration, as described in the preceding paragraph.

Our findings on microbial communities in the case of Portland cement agree with the findings by Wang et al. [27] and Okabe et al. [28]. They reported the effects of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) in the biodeterioration of concrete sewage sewers. SRB (e.g., Desulfobulbus spp.) initiates the deterioration process during anaerobic sewage treatment of high-sulfate-containing domestic wastewater by reducing the sulfate and oxidizing biodegradable organic matter to produce H₂S and CO₂ (Equation (1)) [29,30]. Then, the produced H₂S is oxidized to H₂SO₄ by SOB (e.g., Thiobacillus spp. and Halothiobacillus spp.) in the oxic zone (Equation (2)) [31]. However, other SOB bacteria are also involved in sulfide oxidation (e.g., Thiothrix spp., Sulfuricurvum spp, Sulfurimonas spp., and Bosea spp.), as reported in our study (Table 3). The produced H₂SO₄ is responsible for the pH decrease in the hydrated cement [3]. As time progresses, the produced acid penetrates inside the cement matrix and reacts with alkaline phases, i.e., portlandite (Ca(OH)₂, and C-S-H), to form secondary minerals, mainly gypsum (CaSO₄), ettringite, and monosulfoaluminate (Ca₄Al₂(SO₄)(OH)₁₂·6H₂O) (Equations (3)–(7)) [13], whereas the produced CO₂ dissolves into water to form calcite (Equation (4) and (5)). Due to these minerals’ significant volume expansion compared to hydrated minerals (e.g., portlandite and C-S-H), they result in crack formation and mass loss; thus, the strength of the cement decreases [13,32]. In the present study, the mass loss (i.e., the difference between reference and exposed samples) in cement specimens increased from 0.3 g (for PC at 10 days) to 1.1 g (PC at 75 days) and then decreased to 0.1 g (PC at 240 days of sewage exposure) (Table 4). The mass loss of the specimens consistently increased from the start of exposure (i.e., 10 days) to 75 days and decreased thereafter (Table 4). This was likely due to the decreased overall abundances of SRB, SOB, and fermenting bacteria, thereby inducing less biodeterioration. For example, the abundance of these bacteria decreased from 50.6% at 75 days to 26.6% after 240 days in PC, whereas in CSAC, it decreased from 50.04% (after 75 days) to 34.9% after 240 days (Table 3). This was further confirmed by the low diversity indices of the microbial communities on the specimens after 240 days, i.e., 3.55 (PC) and 3.77 (CSAC) (Figure 2). Furthermore, the compressive strength of the specimens showed an overall decrease from 68.8 MPa for the reference to 50.2 MPa for specimens after 240 days of sewage exposure (Figure 4). In some instances, strength gains increased, corresponding to slight mass gains for PC specimens at 30 days and 75 days of exposure (Figure 4). These gains in compressive strength were due to hydration processes resulting in further formation of hydrated minerals (portlandite, C-S-H), as shown in the XRD results (Figure 4). Moreover, the gains in strength and mass may have also been due to the diffusion of clay particles present in sewage into the matrix of the hydrating cement [14].

Biochemical reactions involving SRB and SOB bacteria:

OrgC + SO₄²⁻ → H₂S_↑ + CO_2↑ + H₂O

(1)

H₂S + O₂ → H₂SO₄

(2)

Biogeochemical reactions involving precipitation of secondary minerals: gypsum, calcite, and ettringite:

Ca(OH)₂ + H₂SO₄ → CaSO₄·2H₂O (gypsum)

(3)

H₂O + CO₂ → HCO₃⁻ (bicarbonate)

(4)

HCO₃⁻ + Ca(OH)₂ → CaCO₃ (calcite)

(5)

CaSO₄·2H₂O + 3CaO.Al₂O₃ + 26H₂O → Ca₆Al₂(SO₄)₃(OH)₁₂·26H₂O (ettringite)

(6)

Ca₆Al₂(SO₄)₃(OH)₁₂·26H₂O + 2(Ca₃Al₂O₆) + 4H₂O → Ca₄Al₂(SO₄)(OH)₁₂·6H₂O
(monosulfoaluminate)

(7)

The deterioration mechanisms discussed above (and illustrated in Equations (1)–(7)) are mainly influenced by sulfate-reducing and sulfide-oxidizing bacteria. However, in the present study, we found that fermentative and other organic acid-producing bacteria, e.g., Trichococcus spp., Acetobacterium spp., and Propionivibrio spp. (Table 3), also play a role in the biodeterioration of cement-based materials in WWTPs (Figure 6, Figure 7 and Figure 8). The organic acids produced by these bacteria include lactic acid (CH₃CH(OH)COOH), acetic acid (CH₃COOH), butyric acid (C₃H₃COOH), and propionic acid (C₂H₅COOH), along with CO₂ [33,34]. Similar findings were also reported in the previous study during the exposure of concrete to a biogas digester [35]. Organic acids can penetrate the concrete matrix and react with portlandite (Ca(OH)₂) to form calcium salts, e.g., calcium acetate, calcium lactate, or calcium butyrate (Equations (8) and (9)), which weakens the concrete structure [34,35]. The presence of calcium-bearing precipitate was observed in SEM images in our findings (Figure 5) for PC specimens after 240 days of sewage exposure. Furthermore, CO₂ generated along with fermentation products contributed to the carbonation process by forming secondary calcite [36]. The microbial-induced carbonate precipitation has been observed in previous studies [37,38,39], and in many instances, it was associated with mass loss of the concrete, which was also observed in our study (Figure 8, Table 4). Its formation involves bacterial cells taking up Ca²⁺ ions from the surroundings, followed by a reaction of this Ca²⁺ on bacterial cells with incoming carbonate ions (CO₃²⁻) to precipitate calcite (CaCO₃) [38]. The formed calcite forms parts of the cement’s mineralogy, resulting in mass gains or decreases in strength. However, the mineralogical composition of the PC specimens (Figure 4) showed that calcite mineral was present in both the reference and exposed specimens, suggesting that not only are microorganisms responsible for CaCO₃ precipitation, but also, CO₂ from the atmosphere contributes significantly during concrete carbonation.

Biogeochemical reactions between organic acids (e.g., acetic and lactic acids) and cement alkaline minerals (e.g., portlandite) to form calcium salt precipitates:

Ca(OH)₂ + 2CH₃COOH → Ca(CH₃COO)₂·H₂O + H₂O

(8)

Ca(OH)₂ + 2C₂H₄(OH)COOH + 3H₂O → Ca(C₂H₄(OH)COO)₂·5H₂O

(9)

The results of the principal component analysis (PCA) and nonmetric multidimensional analysis (NMDS) (Figure 6 and Figure 7) showed that different PC specimens were separated from each other, suggesting a change in the microbial composition during the different exposure periods. This can be attributed to the mineralogical composition of the specimens (Figure 4) as well as connected to the maturing biofilm. The Pearson correlation analysis (Figure 8) showed significant relationships between minerals and bacterial genera: calcite and Acetobacterium spp. (p = 0.02, R² = 0.98), alite and Desulfomicrobium spp. (p = 0.005, R² = 0.97), as well as alite and Sulfurospirillum spp. (p = 0.02, R² = −0.97), suggesting that the individual bacterial communities influenced the increases or decreases in these minerals and vice versa, as has also been reported in the literature [40]. For example, the proliferation of acid-producing microorganisms may reduce the pH and cause the dissolution of alkaline minerals like portlandite [10]. In the present study, all bacterial groups that had a positive correlation with calcite also showed a positive relationship with the mass loss of the specimens (Figure 8), suggesting that the calcite formation is also responsible for the mass loss. This agreed with the previous study by Kong et al. [41]. However, studies [42,43], have reported the positive effects of carbonation (e.g., in soil stabilization) and concrete self-healing, where calcite is responsible for filling up cracks and improving mechanical properties, elsewhere.

As an alternative to Portland-based cement, several new cement materials, including calcium sulfoaluminate cement (CSAC) [15], have emerged in applications affected by microbial-induced corrosion, such as in WWTP facilities. In the literature, different properties have been mentioned that make CSAC resistant to microbial-induced corrosion: (a) absence of alkaline minerals, i.e., portlandite-Ca(OH)₂ and C-S-H), which are attacked easily by biogenic H₂SO₄ [18]; (b) presence of gibbsite (Al(OH)₃) that can release Al³⁺ into porewater solution and acts as an antimicrobial agent [44]; and (c) early formation of ettringite as the hydration product. Our findings showed that most microbial communities (i.e., SOB, SRB, fermenters, and methanogenic archaea) in the PC specimens were also present in CSAC specimens, suggesting that microorganisms strongly colonize CSAC surfaces (Figure 2 and Figure 3). Moreover, our study observed no significant deterioration effects in CSAC specimens due to their stable mineralogical compositions dominated by ettringite (Figure 4). The ettringite observed in CSAC specimens (Figure 4b) was due to the hydration reaction of the clinker minerals (e.g., ye’elimite, anhydrite), whereas that in PC specimens (Figure 4a) was due to the deterioration process as a result of the sulfate reaction. The ettringite in CSAC is responsible for early strength development, fast hardening, and shrinkage compensation [45]. These characteristics, together with the stability of the mineral (Figure 4) and fewer microstructural changes compared to PC (Figure 5), improve the performance of CSAC in sewage applications and other areas affected by microbial-induced deterioration. However, the micro-cracks observed in the CSAC specimens after 75 and 240 days (Figure 5), as seen in the present study, are the result of early ettringite formation, as reported in the literature [43], and not the effects of the deterioration process, as in the case of PC.

The effects of microorganisms on the biodeterioration of cement-based materials in wastewater treatment plants depend on the maturation of the biofilm layer. This layer comprises a consortium of microorganisms, such as bacteria, fungi, and algae, embedded in a self-produced matrix of extracellular polymeric substances (EPS) [10]. The maturation of this layer depends on the following: (a) the difference in microbial community composition and abundance in the biofilm developed on different cement types, as observed in the present study (Figure 2 and Figure 3). For example, after 75 days of sewage exposure, the relative abundance of Desulfomicrobium spp. was 18.3% in PC and 11.3% in CSAC at the same time of exposure (Table 3). Low bacterial abundances in CSAC specimens are due to the presence of gibbsite (Al(OH)₃) and high pH [16]. (b) Surface pH is another factor that influences the biofilm of the material. In the present study, it was observed that the change in the pH values of cement specimens corresponded to the total relative abundance of degradation-related bacteria (e.g., SRB, SOB and fermenters) (Table 3), suggesting a relationship between surface pH and microbial adhesion on the surface of cement specimens. The first adhesion was conducted by autotrophic bacteria (i.e., photoautotrophs and chemoautotrophs), followed by the interaction of planktonic or free-floating microorganisms with the formed biofilm. When the thickness of the biofilm increased, the anaerobic bacteria predominated underneath parts of the biofilm close to the concrete surface, whereas the aerobic bacteria remained on the outer parts adjacent to the water [46]. The last phases are the maturation and dispersion phases, which involve massive cell growth due to efficient diffusion of nutrients (e.g., organic matter, O₂, SO₄²⁻, NO₃⁻) [47] and detachment of the loosely bound cell, which separates and adheres elsewhere in the system. (c) The physical properties of the surface (e.g., porosity and roughness) also influence microbial adhesion. Because of their heterogeneous nature, cement-based materials are characterized by high porosity and roughness. These properties provide a large surface area and microenvironments for more microbial adhesion. As a result, rough and porous surfaces have more microbial adhesion and high biodeterioration as compared to less porous and rough surfaces [48].

5. Conclusions

This paper studied the microbial-induced corrosion of Portland cement (PC) and calcium sulfoaluminate cement (CSAC) during sewage treatment in a wastewater treatment plant. Our results showed that the PC specimens exhibited higher deterioration effects than the CSAC specimens with similar exposure periods. The main deterioration effects observed in the studied specimens were (a) mass loss (up to 2.63%) in PC and (2.86%) in CSAC, (b) pH reduction (from 8.95 to 7.75%) in PC and (9.68 to 8.84) in CSAC, and (c) secondary minerals (e.g., calcite). The ratio of the bacteria genera associated with cement biodeterioration (i.e., SRB, SOB and fermenting bacteria) was up to 50% of the total bacteria communities in the specimens after 240 days and was significantly correlated with PC characteristics (calcite and mass loss). In contrast, no significant correlations were observed between CSAC characteristics and microbial communities. Owing to its stable characteristics related to biodeterioration processes, CSA-based cement is offered to construct WWTP facilities in order to overcome the drawbacks discussed regarding PC materials. This cement can also be blended with PC to improve its characteristics and increase the long-term durability of WWTP facilities.