Characterization of sulfur oxidizing bacteria related to biogenic sulfuric acid corrosion in sludge digesters
© The Author(s). 2016
Received: 28 February 2016
Accepted: 8 July 2016
Published: 18 July 2016
Biogenic sulfuric acid (BSA) corrosion damages sewerage and wastewater treatment facilities but is not well investigated in sludge digesters. Sulfur/sulfide oxidizing bacteria (SOB) oxidize sulfur compounds to sulfuric acid, inducing BSA corrosion. To obtain more information on BSA corrosion in sludge digesters, microbial communities from six different, BSA-damaged, digesters were analyzed using culture dependent methods and subsequent polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). BSA production was determined in laboratory scale systems with mixed and pure cultures, and in-situ with concrete specimens from the digester headspace and sludge zones.
The SOB Acidithiobacillus thiooxidans, Thiomonas intermedia, and Thiomonas perometabolis were cultivated and compared to PCR-DGGE results, revealing the presence of additional acidophilic and neutrophilic SOB. Sulfate concentrations of 10–87 mmol/L after 6–21 days of incubation (final pH 1.0–2.0) in mixed cultures, and up to 433 mmol/L after 42 days (final pH <1.0) in pure A. thiooxidans cultures showed huge sulfuric acid production potentials. Additionally, elevated sulfate concentrations in the corroded concrete of the digester headspace in contrast to the concrete of the sludge zone indicated biological sulfur/sulfide oxidation.
The presence of SOB and confirmation of their sulfuric acid production under laboratory conditions reveal that these organisms might contribute to BSA corrosion within sludge digesters. Elevated sulfate concentrations on the corroded concrete wall in the digester headspace (compared to the sludge zone) further indicate biological sulfur/sulfide oxidation in-situ. For the first time, SOB presence and activity is directly relatable to BSA corrosion in sludge digesters.
KeywordsAcid attack Concrete corrosion Sludge digester Sulfur oxidizing bacteria Thiobacillus Biogenic sulfuric acid Sulfate reducing organisms
Microbial deterioration of concrete by biogenic sulfuric acid (BSA) is a serious and common problem in wastewater treatment facilities. Worldwide, maintenance and retrofitting of degraded concrete structures costs several billions of dollars every year . BSA corrosion is a multistage process of sulfur/sulfate reducing (SRB) and sulfur/sulfide oxidizing bacteria (SOB). The first, anaerobic, step occurs when SRB reduce sulfate and other oxidized sulfur compounds to hydrogen sulfide (H2S) . H2S volatilizes and dissolves in the moist concrete surface . The initial pH of concrete is approximately 12.0, a value hardly allowing microbial growth . H2S, CO2 and other gases with acidic properties abiotically decrease the pH to values around 9.0 enabling the colonization of neutrophilic sulfur oxidizing bacteria (NSOB) such as Thiobacillus spp. and Thiomonas spp. . These NSOB, oxidize H2S and other reduced sulfur compounds to sulfuric acid (H2SO4) and polythionic acids thus lowering the pH to around 3.5–5.0 . At pH 5.0 and below, acidophilic sulfur oxidizing bacteria (ASOB) such as Acidithiobacillus thiooxidans, continue sulfur oxidation by producing high amounts of sulfuric acid that decreases the pH to 1.0–2.0 [6, 7]. H2SO4 reacts with the cement matrix leading to the formation of gypsum (CaSO4 · 2H 2 O) and ettringite (3CaO · Al2O3 · 3CaSO4 · 32H2O) . These expansive sulfate salts lead to internal cracks in the concrete and finally to structural failure . Corrosion rates of several millimeters per year are reported for sewer pipes . BSA corrosion, although well described in sewer pipes, is hardly investigated in sludge digesters where anaerobic conditions enable the growth of SRB and H2S production [11, 12], but the occurrence of aerobic SOB, comes unexpected.
Design characteristics of the six digesters A-F
Year of construction
Digester volume [m3]
Operating temperature [°C]
Sulfate in drilling dust headspace [% w/w]
Sulfate in drilling dust sludge zone [% w/w]
SOB enrichment and cultivation
PCR amplification of the 16S rRNA gene
For the identification of SOB pure cultures, colony PCRs were carried out using universal bacterial primers 27f (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492r (5′-TAC GGY TAC CTT GTT ACG ACT T-3′ ) amplifying the nearly full-length 16S rRNA gene. For DGGE analysis of the mixed SOB cultures, a 16S rRNA gene fragment (~550 bp) was amplified using bacterial primers 27f and 517r (5′-GTA TTA CCG CGG CTG CTG GC-3′ ), with the forward primer containing a GC-clamp (40 bp) at the 5′end (5′-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCG CCC CCG CCC CGG-3′ ). PCR conditions for the primers 27f/1492r and 27f/517r included an initial denaturation at 95 °C for 2 min, 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 95 °C for 30 s and final elongation at 72 °C for 5 min. PCR primers were obtained from Eurofins MWG Operon (Ebersberg, Germany). PCR was carried out with a primus 96 cycler (PeqLab Biotechnologie GmbH, Erlangen, Germany) using GoTaq(R) G2 Hot Start Colorless Master Mix (Promega GmbH, Mannheim, Germany) according to manufacturer’s instructions.
SOB-diversity studies in mixed cultures were performed with DGGE using 15 μl of the 550 bp PCR product. Separation was carried out with a 6 % (w/v) polyacrylamide gel using the DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, Munich, Germany). A denaturing gradient from 20 to 80 % was used (100 % denaturing solution defined as 7 M urea and 40 % (v/v) formamide). Electrophoresis was performed at 55 °C for 16.5 h at a constant voltage of 60 V. The polyacrylamide gels were stained with ethidium bromide (0.5 μg/mL) for 20 min, rinsed with Milli-Q-water (Millipore, Bedford, USA), documented under UV-light (312 nm), and the dominant bands cut out with a sterile scalpel. The DNA was eluted in sterile Milli-Q water (24 h, 37 °C) and re-amplified using the primers 27f and 517r without GC-clamp.
16S rRNA sequencing
Purified PCR products (innuPREP DOUBLEpure Kit, Analytik Jena, Jena, Germany) were sequenced by Eurofins MWG Operon (Ebersberg, Germany). Sequences were assembled with Geneious 7.1.7 (http://www.geneious.com), analyzed with ENA (European Nucleotide Archive) sequence search (http://www.ebi.ac.uk/ena/search), and aligned with SINA 1.2.11 . Phylogenetic analyses were performed with MEGA6 . Phylogenetic trees of nearly full length and 16S rRNA gene fragments were calculated based on the maximum composite likelihood method with 2,000 bootstrap replications (n = 2,000). Sequences obtained from pure cultures and DGGE bands were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena) to get accession numbers.
SOB activity of mixed and pure cultures
pH and sulfate concentration measurements
Incubation time [d]
Final sulfate concentration [mmol/L]
A long-term sulfate measurement for 42 days in batch-configuration (DSMZ medium 35), monitored the BSA production of isolate DgE-1 (99 % similarity with A. thiooxidans, LN864656).
In-situ SOB activity monitoring
To gain information about the SOB activity in-situ, the sulfate content on the digester concrete wall was determined (Table 1). One concrete composite sample consisting of three bores was taken from the headspace and the sludge zone from every digester showing characteristic corrosion damage patterns (washed out concrete surface). The concrete was sampled in form of drilling dust in a depth of 0–40 mm using a hollow drill (25 mm diameter) by Weber-Ingenieure GmbH (Pforzheim, Germany). For sulfate measurements, the drilling dust samples were thermally disintegrated at 80 °C and 15 % (v/v) hydrochloric acid followed by a photometrical analysis at 436 nm (Nanocholor 500 D, Macherey und Nagel, Germany). The analysis was performed by the laboratory “Dr. Michael Figgemeier-Baustoffanalyse & Bauphysik” (Ludwigsburg, Germany).
Enriched SOB cultures
Pure SOB cultures
Enrichments that exhibited a significant pH decrease were streaked on their corresponding solid medium to isolate pure SOB. For isolation, DSMZ medium 68 (pH 6.0) and Thiobacillus medium (pH 4.1) showed the best results. Approximately 40 isolates that showed a pH decline on the agar medium (visible due to pH indicators) were identified by 16S rRNA gene sequence analysis. Three sulfur oxidizing species, associated with concrete corrosion, were closely related to Acidithiobacillus thiooxidans, Thiomonas intermedia and Thiomonas perometabolis. Their phylogenetic relation is displayed in Fig. 4 . In Dg A and E, the acidophilic Acidithiobacillus thiooxidans. and neutrophilic Thiomonas spp. were detected, whereas in Dg B, D and F only neutrophilic Thiomonas spp. were identified.
By applying different media, varying in initial pH and sulfur components, a variety of SOB species detected by DGGE in mixed culture (Fig. 3) was obtained. Enrichment and cultivation of mixed cultures worked best in DSMZ medium 68 and Thiobacillus medium. For cultivation of pure A. thiooxidans, best growth occurred in DSMZ medium 35 (pH 4.5) with elemental sulfur as sole energy source. After an incubation period of two weeks, A. thiooxidans reduced the pH in DSMZ medium 35 from 4.5 to 0.5 indicating a high sulfuric acid production and BSA corrosion potential. For cultivation of pure Thiomonas spp., media with Na2S2O3 and initial pH values of 4.0–6.0 showed best results (DSMZ 68 and Thiobacillus medium). Thiomonas spp. reduced the pH within the used DSMZ medium 68 from 6.0 to 2.5 after two weeks of incubation, also revealing a high acid production potential.
In-situ BSA activity in sludge digester
All analyzed digesters (A-C and E-F) showed a higher sulfate content on the concrete surface of the digester headspace than in the sludge zone (Table 1). The highest difference was observed in Dg A, where the sulfate concentration in the headspace (1.2 % w/w) was more than ten-times higher than in the sludge zone (0.1 % w/w), potentially resulting from the activity of the ASOB A. thiooxidans found in this digester (Fig. 4).
Cultivation and isolation of active SOB communities
SOB within the biofilm samples were specifically enriched to test their sulfuric acid production activity. Although cultivation dependent techniques may not be appropriate to draw a comprehensive picture of the microbial community, they are the only and still powerful method to investigate the capability of sulfuric acid production and BSA corrosion potential under defined conditions. By enrichment in specific media, different SOB species were purified (Fig. 4). A variety of BSA related bacteria were identified in the liquid cultures (Fig. 3), and their sulfuric acid production capacity was demonstrated under laboratory conditions (10–433 mmol/L).
For SOB purification different media with elemental sulfur and sodium thiosulfate as only energy sources were applied to obtain a high SOB diversity. Thiosulfate, the most frequently used substrate for SOB cultivation, is, in contrast to elemental sulfur, highly water soluble and stable over a broad pH range . In addition, autotrophic media were utilized as the main contributors to corrosion, e.g., Acidithiobacillus spp. and Thiomonas spp., are known obligate or facultative autotrophs. Furthermore, such media suppress the growth of unwanted heterotrophic bacteria, which are most likely dominant in the biofilm sample. One major problem in obtaining selective enrichment of chemolithoautotrophic organisms is the contamination through organic compounds  resulting in the detection of non-SOB species. Sources for heterotrophic contaminants are i) contaminated water or chemicals used for media preparation, ii) trace amounts of soluble organic material in almost all agar brands, and iii) secretion of organic compounds by obligate chemolithotrophs . Apart from the identification of a few heterotrophic non-SOB species, the application of selective culture media allowed to specifically enrich the organisms of interest (Figs. 3 and 4). This shows the potential of highly selective media as the desired organisms grew best under these conditions. However, a more comprehensive picture of the SOB diversity within the liquid media was drawn by PCR-DGGE (Fig. 3). It has to be mentioned, that only the combination of cultivation dependent and -independent (PCR-DGGE) techniques revealed a variety of taxonomically different sulfur oxidizers that can be classified in acidophilic and neutrophilic sulfur oxidizing bacteria (ASOB and NSOB) as well as non-SOB.
Acidophilic sulfur oxidizing bacteria (ASOB)
Acidithiobacillus spp. and/or Alicyclobacillus sp. were identified in enriched cultures of Dg A, B, D and E (Figs. 3 and 4) and are known to produce sulfuric acid from reduced sulfur compounds . The identification of ASOB in the apparently neutral digester environment suggests that pH gradients and “acidic microniches” might be present, especially in the biofilm of the digester headspace .
All members of the genus Acidithiobacillus are obligate acidophiles and characterized by chemolithoautotrophic growth . Pure A. thiooxidans with a growth optimum at pH 2.0–4.0 can be cultivated in acidic media with elemental sulfur as the only nutrient [20, 22], as has been confirmed in this study as well. A. thiooxidans, found in Dg A, D and E, is a key organism for BSA corrosion, because it has been the most dominant species in heavily corroded concrete samples [3, 6]. A. thiooxidans can produce high amounts of sulfuric acid and grows at pH values as low as 0.5 [6, 20]. In this study, A. thiooxidans produced a sulfuric acid concentration of 4 % (Fig. 5). Cwalina  stated that biogenic H2SO4 in concrete pores may even reach 10 %.
Alicyclobacillus sp. was detected within the enrichment cultures of Dg D and E using PCR-DGGE. A few members of the genus Alicyclobacillus have been described as sulfur- and ferrous-oxidizing . A study by Vupputuri et al. , analyzing the microbial diversity on concrete surfaces from deteriorated bridge structures, revealed that Alicyclobacillus spp. was the most dominant sulfur oxidizing acid producer that reduced the pH value of the culture medium from 6.7 to 2.8.
Neutrophilic sulfur oxidizing bacteria (NSOB)
The presence of Thiomonas intermedia and Thiomonas perometabolis, obtained in pure (Fig. 4), was already described in corroded concrete samples [3, 26, 27]. A study by Wei et al.  using liquid cultures inoculated with corroded material from a bridge support, found T. perometabolis as the dominant acid producer.
Another NSOB, Paracoccus sp., occurred in liquid cultures of Dg A and C and is known to oxidize reduced sulfur compounds (e.g., thiosulfate and elemental sulfur) to generate energy for autotrophic growth .
The genera Ancylobacter, Mesorhizobium, Hyphomicrobium and Delftia comprise sulfur/sulfide oxidizing species, but are not typically mentioned in the context of BSA corrosion. Growth tests with Ancylobacter aquaticus showed its ability to grow chemolithoautrotrophically when thiosulfate was provided as only energy source . For Mesorhizobium thiogangeticum, originally identified in rhizosphere soil, chemolithoautotrophic growth was observed with Na2S2O3 and S0 . Hyphomicrobium sp. is known for its oxidation of hydrogen sulfide to elemental sulfur . SOB, isolated from a rice field soil, were closely related to Delftia sp.  indicating its ability for sulfur-oxidation.
Other heterotrophic microorganisms not commonly associated with sulfur oxidation and thus termed non-SOB, e.g., Sphingobacterium sp., Sphingomonas sp., and Stenotrophomonas sp., were detected in this study as well. The identification of heterotrophic non-SOB in the enrichment cultures was probably due to their presence in the original biofilm. A contamination with organic residues from the biofilm sample may have enabled the growth of heterotrophic non-SOB in the culture media. Furthermore, many obligate chemolithotrophic sulfur oxidizers produce organic substances that could be subsequently utilized by heterotrophs  leading to the growth of non-SOB species. However, the non-SOB detected in this study might still play an important role because their presence was already reported in several samples of corroded concrete originating from different sewer pipes. SOB might interact with non-SOB in the biofilm matrix where excreted metabolites could serve as nutrients for non-SOB or vice versa. Sphingobacteriales, for instance, are dominant in microbial induced concrete corrosion layers  and Sphingomonas sp. was detected in corroded sewer pipes above the water level . Stenotrophomonas maltophilia was found in slightly corroded concrete material but also observed in the surrounding of the steel bar [3, 33].
Oxygen availability and BSA corrosion in sludge digesters
In contrast to sewer pipes, oxygen availability in sludge digesters is rather limited [11, 34] and thus, crucial for BSA corrosion. However, in this study typical BSA corrosion damage patterns, characterized by washed out concrete surfaces, were observed in the headspace of several digesters (see Fig. 1), indicating SOB activity resulting in BSA production and corrosion. Thus, oxygen carriers must be at least available in small patches fostering the growth of sulfur-oxidizing communities. Many sulfur oxidizers can grow in niches, where sulfide and oxygen coexist . When oxygen, even at low levels, is available, sulfur oxidizers can spontaneously oxidize sulfide. Very high turnover rates of sulfide were reported even at extremely low concentrations of sulfide and oxygen (< 10−6 mM) . It is supposed that oxygen availability in microniches might be sufficient for SOB activity and enable the oxidation of sulfur compounds to sulfate or sulfuric acid. Thermodynamically, oxidation of sulfuric compounds is always favored compared to the oxidation of methane, as is applied in biological in-situ desulfurization in digesters.
Under anaerobic conditions, sulfate, which is commonly found in sewage sludge , can be reduced to sulfide (S2−) by anaerobic SRB (e.g., Desulfovibrio spp.). S2− can be abiotically or biotically converted by, e.g., Hyphomicrobium spp., to elemental sulfur [31, 35]. Elemental sulfur can either be further oxidized by other SOB species (e.g., Alicyclobacillus spp. , and Acidithiobacillus spp.) in case of oxygen availability, or might be reconverted under anaerobic conditions to sulfide by SRB such as Geobacter spp. The concurrent detection of SRB and SOB in sludge digesters  indicates that these two groups might interact to oxidize and reduce sulfur compounds. Increased sulfate concentrations on the concrete wall of the digester headspace (compared to the sludge zone) provided further evidence for biological sulfur oxidation in the headspace. However, only a steady sulfur/sulfide oxidation over years to decades could result in the characteristic corrosion damage pattern as shown in Fig. 1.
This study revealed the presence of different SOB species on the headspace concrete wall of six sludge digesters and showed their capability to produce BSA under laboratory conditions. The identified SOB species potentially contribute to BSA corrosion in sludge digesters, especially as elevated sulfate concentrations on the concrete walls of the digester headspace were measured in-situ. However, further investigations on the availability of oxygen carriers, sulfide turnover rates and SOB activity in digester systems are vital to finally draw a conclusive picture about the BSA production in situ.
The authors would like to thank Weber-Ingenieure GmbH for providing pictures of the digester headspace, biofilm and drilling dust samples. Lisa-Marie Rempe is acknowledged for technical assistance.
This study was funded by the Bundesministerium für Wirtschaft und Energie (BMWi), Zentrales Innovationsprogramm Mittelstand, ZIM-Kooperationsprojekt (grant number KF2990501SA2). This work was supported by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program.
BeH drafted the manuscript, designed and carried out the biodegradation experiments. BH, JD, and KK reviewed and edited the manuscript. EM conceived of the study and helped to review the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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