Alejandra Carolina Zamora*, Nora Malaver

 Laboratorio de Microbiología Ambiental, Instituto de Zoología y Ecología Tropical, Facultad de Ciencias, Universidad Central de Venezuela. Caracas, Venezuela.

* Correspondencia:E-mail: alejandra.zamora@gmail.com

Abstract: This paper presents a comparison of various methods for culture, quantification, and maintenance of sulfate-reducing bacteria (SRB) under laboratory conditions, using liquid and semisolid media for water and soil samples. Starkey, Postgate B, API and modified Baars media were used with an incubation time of 21 days in a GasPack™ anaerobic jars type. The modified Baars medium was more efficient for the quantification of SRB in both liquid and semisolid media when compared with other culture media tested, detecting differences of three orders of magnitude in soil samples and in two orders for water samples at 8 days of incubation. The semisolid modified Baars medium in Petri dishes allowed the isolation of pure cultures of SRB by the streak plate method. It was found that strains in liquid modified Baars medium remain viable for up to three months, while in the same semisolid medium were kept only one month.

Keywords: hydrogen sulfide, sulfate-reducing bacteria, culture media, culture methods, SRB quantification.

Aspectos metodológicos para el cultivo y la cuantificación de bacterias heterotróficas sulfato-reductoras

Resumen: Este trabajo presenta una comparación de diversos métodos para el cultivo, cuantificación, y mantenimiento de bacterias sulfato-reductoras (BSR) en condiciones de laboratorio, utilizando medios líquidos y semisólidos para muestras de agua y suelo. Se utilizaron los medios de cultivo de Starkey, Postgate B, API y Baars modificado con un tiempo de incubación de 21 días en jarras de anaerobiosis tipo GasPack™. Se determinó que para la cuantificación de SRB, tanto en medio líquido como en semisólido, el medio Baars modificado es más eficiente comparado con los demás medios de cultivo probados, detectando diferencias de tres órdenes de magnitud en muestras de suelo y de dos órdenes de magnitud en muestras de agua a los 8 días de incubación. El medio Baars modificado semisólido servido en placas de Petri permitió el aislamiento de cultivos puros de BSR mediante siembra por agotamiento. Se encontró que en el medio modificado de Baars líquido las cepas se mantienen viables hasta por tres meses mientras que en el mismo medio semisólido sólo se mantienen durante un mes.

Palabras clave: sulfuro de hidrógeno, bacterias sulfato-reductoras, medios de cultivo, métodos de cultivo, cuantificación de SRB.

Recibido 21 de noviembre de 2011; aceptado 20 de mayo de 2012

The sulfate-reducing bacteria (SRB) are strict anaerobic microorganisms; however, they can be found in aerobic environments, specifically in anoxic micro-zones [1]. They constitute a diverse morphological group including cellular forms such as cocci, bacillus, cellular aggregates and multi-cellular filaments.

The heterotrophic SRB obtain their energy for cell synthesis and growth from oxidation of organic compounds. This process is coupled to the reduction of sulfate to hydrogen sulfide, through several metabolic pathways, but the most common in natural environments is the dissimilatory reduction of sulfate to sulfide. This sulfide can be released as hydrogen sulfide or precipitate in the soil as ferrous sulfide (FeS) in a process linked to the oxidation of organic matter [2,3].

The hydrogen sulfide (H₂S) released by the SRB is a strong reducing agent, highly toxic to other organisms. The SRB have economic importance for the industries, due to corrosion and contamination problems that cause the production of H₂S [4,5]. The H₂S reacts with water to form sulfuric acid, which deteriorates the metal pipes and concrete structures [6,7] in a process known as microbiologically influenced corrosion (MIC) [8]. The MIC occurs due to the aggressive attack of the metabolites produced during the adhesion of microorganisms to the metal (pitting corrosion) [9] or concrete surfaces (biocorrosion).

However, the activity of the SRB has great ecological importance because of their involvement in the mineralization of organic matter in anaerobic environments [10]. The SRB are responsible for degrading about 53% of the organic matter in marine and coastal environments, where sediments have low redox potential and high sulfate content, limiting the oxidation of organic substrates [11]. They also have been used in bioremediation of toxic pollutants to the environment, such as toluene and xylene [12]. This process is possible because the SRB are capable of breaking the ring structure of some organic compounds, and consequently, these are mineralized [13].

Therefore, the development of techniques for detection, quantification and isolation are necessary to understand the role of SRB in different industrial and ecological processes. Considerable efforts have been oriented to develop fast and reliable methodologies for detection and quantification of SRB in natural and artificial environments [14]. In general, the methods used to enumerate these bacteria can be divided in two different categories: (i) direct detection and (ii) culture methods.

Direct detection of SRB is mostly used in current research involving these bacteria. This method has been developed in the last few years and includes various techniques such as the use of cultivate antibodies of SRB [15], immunodetection of the enzyme adenosine-5’-phosphosulfate (APS) reductase [16] and sequence analysis of genes encoding 16S rRNA [17,18], particularly the analysis of the dissimilatory sulfite reductase genes (dsrAB) [19].

Culture methods for quantification of SRB based on the most probable number (MPN) technique has been widely used for a long time [5,10,17,20-22]. Up to this point, a great variety of culture media have been developed for specific environments, including activated sludge, marine sediments and samples from the oil industry [1,2,5,10,20,22,23]. All these culture media contain lactate as carbon and energy source, and the presence of SRB is always assessed by the formation of a black color precipitate of FeS.

However, traditional techniques based on selective microbial cultures offer a limited utility for quantification and characterization of the SRB isolated from environmental samples. Most strains do not grow up in vitro either because the culture media do not have the specific growth conditions or, in other cases, different strains of SRB are interdependent with other microorganisms [24]. For this reason, the number of SRB detected by culture methods may be underestimated [25]. Thus, the isolation of pure cultures and maintenance of the strains in the laboratory are very complicated tasks. They demand great efforts and time because these bacteria lose viability in a short time.

Real-time PCR is a sensitive and rapid molecular method, which permits detection and quantification of bacterial populations through DNA technology. This approach does not require culture of the target organisms and is therefore ideally suitable to studies [25,26]. However, these techniques involve high costs and specialized equipment, often not available if the studies are not associated with scientific research.

Materials and methods

Specimen collection: It were used two specimen types: (a) a water specimen taken from a refinery effluent, intended to come into contact with hydrocarbons; (b) a sediment specimen collected by obtaining three cores from shallow permanently water-covered soil (approximately 10 cm depth) from the Lower Orinoco region, Anzoátegui State, Venezuela. These samples were stored in sterile containers at 4 °C until processing in the laboratory.

SRB activation in enriched cultures: To detect the presence of SRB, collected samples were previously activated in Starkey liquid medium as described by Toerien et al. [20]. This medium was prepared by initially heating the components and subsequently 95 mL was served in bottles under a N₂ atmosphere and hermetically sealed. It was sterilized in an autoclave at 121 ºC with 15 pounds per square inch of pressure for 20 minutes. Five (5) mL of the water specimen were directly inoculated in the medium using a sterile syringe. For the determination of SRB in the sediment, 10 gr of the sediment were placed inside an Erlenmeyer containing 90 mL of 0.85% NaCl solution and a paraffin layer of 3 mm to create anaerobic conditions. The Erlenmeyers were shaked during 8 hours for the SRB activation and 5 mL of this solution were inoculated into the bottles containing Starkey liquid medium. Bottles were incubated in darkness for 21 days at 25 ºC. The presence of SRB was determined by the formation of a FeS black precipitate on the bottom of the bottle.

Enumeration of SRB: The enumeration of SRB was performed using the Baars’s modified medium [27], in which the calcium sulfate was substituted by sodium sulfate and calcium chloride, and a 50% sodium lactate solution was used instead of a 70% solution as a carbon source (Table 1). The medium was prepared by dissolving the salts in distilled water and autoclaving for 20 minutes at 121 ºC with 15 pounds per square inch of pressure. Separately, the complementary solution was prepared and sterilized by filtration (Millipore^® membrane HA type) and was added to the medium base at 5% (v/v). The semisolid medium was prepared adding 8 gr. l^-1 of purified agar (55% w/v) in aerobic conditions.

Table 1. Base medium used for enumeration and isolation of pure cultures of sulfate-reducing bacteria(*).

(*) Baars´s modified medium (1930). (†) The original Baars´s medium contains calcium sulfate at a ratio of 1 g/L. ‡ The Baars´s medium (1930) contains sodium lactate 70%. (§) For semisolid medium.

To compare the efficiency of the tested culture medium, the quantification of SRB was carried out in Postgate´s B and Starkey´s liquid and semisolid media [21,22] as well as in API medium (Himedia ®) incubated under the same conditions.

SBR isolation from pure cultures of SRB: The isolation and purification of the strains was performed following the streak plate method directly from growth on the agar surface, using Baars´s modified semisolid medium [27].

Statistical analysis: A Student t test was used to determine whether MPN and CFU values obtained with different media were significantly different. Differences were considered significant at the 95% confidence level.

Results and discussion

SRB enumeration in water and sediment specimens: Figure 1 shows the number of SRB quantified in the water specimen. MPN values that fluctuated between 1.8x10³ and 2.4x10⁴ at 8 days of incubation followed the same trend of the CFU in semisolid medium (from 1.6x10³ to 1.6x10⁴) using Baars´s modified medium [27]. Differences of one to two orders of magnitude were found for water specimens (Figure 2) when compared Baars´s modified medium with Postgate´s B and Starkey’s media (Anova, p<0.05).

The SRB density in the API synthetic medium underestimated the number of CFU, since only were detected after 21 days of incubation, showing differences of more than three orders of magnitude compared with the specimens grown on other culture media (Figure 1). It was also found that Starkey and Postgate B semisolid media required an incubation period of 15 days.

A similar pattern was reported by Jain [2] who determined that use Postgate´s B semisolid medium requires less incubation time (7 days) compared with synthetic media. A similar result was obtained by Vester & Ingvorsen [10] who compared different culture media for SRB enumeration through the MPN technique, finding that synthetic media significantly underestimate the SRB number. These saline media containimg lactate as unique electron donor, while the API synthetic medium has yeast extract. Regarding the latter idea, Gibson et al. [28] argued that a large number of SRB natural populations were physiologically different from laboratory strains, and were not able to grow in media containing high levels of organic substrates.

The sediment specimens (Figure 2) showed a SRB concentration which only required 8 days of incubation, whereas the API medium required an incubation time of at least 21 days. The differences in the SRB counts determined on the tested media could be related to the procedures used for activating the sediment into the liquid medium using a 0.85% NaCl solution, because the concentration of salts, particularly the NaCl, should change the bacterial concentration in the specimen being enumerated [29]. Also the absence of a reducing agent in the medium (e.g. cystein, rezarsurin) could be important.

It is interesting to note the presence of sulfate-reducing bacteria in the upper section of sediment. In this aspect, it has been recently demonstrated that SRB were present in oxygenated and non-oxygenated environments, especially those close to surface sediments [30]. This might explain the presence of SRB on sediment and water specimens studied.

The results shown on figures 1 and 2 indicate that the number of viable SRB was greater when using saline mediums (Baars, Postgate B and Starkey) when compared to the synthetic media tested (API). In this regard, Jain [2] showed that with the API medium [23] the values of MPN were significantly lower than those determined by the Postgate B and Baars media. Furthermore, Tanner [29] used API-38 commercial medium suggested by the American Petroleum Institute [23], and determined that SRB density detected by the commercial media was lower compared to Baars and Postgate B media. Moreover, their study found that the addition of ammonium salts, calcium and phosphorus improved the sensitivity of the API medium. According to this author, the data presented on figures 1 and 2 indicate that saline medium were more efficient for the SRB enumeration. Moreover, the number of SRB quantified in this work was similar to that reported by Tanner [29] for sediment specimens at the same incubation time (from 10² to 10³ MPN).

Starkey and Postgate B media did not show any results using conventional anaerobic techniques (GasPak™ jars with anaerobic generator Anaerocult A), whereas when the flasks were gassed with N₂ there were SRB expression. This pattern could be attributed to the culture media preparation conditions and inoculation technique, because these media are frequently used with techniques in which the atmosphere on the incubation chamber is purged of oxygen using anoxic gases, such as N₂ or a mixture of N₂:CO₂ as a redox agent with an 80:20 ratio [31]. In this sense, Tanner [29] suggested that anaerobic conditions and a low redox potential are important for the estimation of SRB, especially from environmental specimens.

It is important to note that in media containing lactate as electron donor, the Desulfovibrio or Desulfococcus groups prevailed in SRB counting [32]. This approach is limited because the SRB diversity is broadest. However, the use of this methodology for preliminary detection on SRB in environmental and industrial samples will enhance the monitoring of these microorganisms.

SRB isolation and maintenance from pure cultures: The Baars´s medium or similar ones have been used a long time for the maintenance of SRB pure culture [26,29]. SRB colonies showed a blackening of the medium allowing for quantification and subsequent isolation using routine microbiological techniques. The strains grown after 4 days of incubation and remained viable up to 30 days in the semisolid medium, whereas in the liquid medium were viable until 90 days.

Another aspect to consider during the isolation of SRB is temperature, because temperature seems to affect the viability of these bacteria. During the tests, it was determined that the platinum loop affects the survival of SRB cells, causing not growth in culture medium. For this reason, sterile wooden sticks were used, resulting in successfully pure strain isolations.

Conclusions

The standardization of a methodology that allows the use of conventional techniques to study SRB are of great applications in microbial ecology.

It has the advantage of being a simple method that requires no special techniques for the preparation of culture media and inoculation, and reduces significantly the incubation time to 8 days compared to other culture media tested. Moreover, Baars´s medium allows the isolation and maintenance of pure viable strains for up to 90 days.

Acknowledgements

The authors thank to Jorge Pérez-Eman for his helpful comments and critical review of this manuscript. We also thank to Susana Marin for improving the English language, and to the three anonymous reviewers who enriched this paper with their critics.

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