Alkalophile microorganisms are defined as organisms that have an optimum growth rate in environments with alkaline pH, particularly greater than 8. A source rich in variety of these organisms are the environments with a stable alkalinity, such as saline lakes (Castillo et al. 2005). Halophile bacteria are defined as microorganisms that show a better growth in the presence of salt NaCl. Given that these microorganisms are capable of growth in different saline concentrations, the term halophile is usually reserved for those requiring a minimum of excess salt concentration, found in seawater, in general, concentrations greater than 3% (Oren, 2008). Extreme halophile bacteria are those that present an optimum growth rate in high saline concentrations, near 20% NaCl (3–4 M). These bacteria grow in hyper saline environments, such as saline lakes that present a greater range of saline concentrations than sea (Jones and Grant 2002). The soil from the Texcoco Lake is alkaline, and with electrical conductivities (EC) in saturated strata from 22 to 150 dSm–1 and sodium percentages from 76% to 98% (Luna-Guido et al. 2002). Several microorganisms adapted to extreme conditions have been identified from the soil of the former Texcoco Lake (Valenzuela-Encinas et al. 2008; Ruiz-Romero et al. 2013; Soto-Padilla et al. 2014). The application of molecular biological techniques to microbial ecology has shown that the cultured organisms are generally different from those that dominate in the natural environment (Oren, 2002). In addition, knowledge of their biochemical characteristics supports understanding of their metabolic processes.
In the last decade, there has been an increment in the number of newly isolated bacterial genera, which grows in alkaline and halophile conditions. The study of enzymes that aid in the metabolism of these extremophiles to operate under these conditions is of great interest (Castillo et al. 2005; Ramírez et al. 2006). Characterization and identification of native microorganisms of relevant sites, as well as specific biological and metabolic functions are closely related to protein and enzymes that work in extreme conditions. These biomolecules show unique features that can be used as models for the design and construction of proteins with new properties, which are of interest for industrial applications (Castillo et al. 2005). The aim of this study was to accomplish biochemical and phylogenetic characterization of the native halophile/alkaline tolerant bacteria from the Texcoco Lake.
The HN31(22) strain was Gram-positive with cells of short roads morphology (Fig. 1) and size between 1–2 μm. Colonies showed a light yellow coloration, with bright creamy and round shape and flat borders. Growth of the bacteria was observed in NaCl at concentrations of up to 20% of (Fig. 2); the most efficient growth rate was observed when 5% NaCl was used, but the bacteria grew also when 10, 15 and up to 20% salt concentrations were employed. With this evidence, the bacterium was considered a halophile. Fig. 3 shows the growth of HN31(22) strain in the evaluated pH values, showing that an optimum growth rate was obtained at a neutral pH of 7. The bacterial multiplication was also observed at pH values of 5 to 10; however, a strong reduction was registered at pH of 11. For this reason the strain was considered as an alkaline tolerant bacteria. Table I shows the growth of the HN31(22) strain in various temperatures.
Growth of bacteria of the HN31(22) strain in marine agar at different temperatures (4, 30, 37, 40, 55°C).
Temperature (°C) | Growth (CFU/ml) |
---|---|
4 | 0 |
30 | 4.2 × 103 |
37 | 34 × 104 |
40 | 18 × 104 |
55 | 0 |
A phylogenetic analysis was carried out and the strain isolated was classified to the genus of
Comparison of phenotypic characteristics of bacteria of the HN31(22) strain with the other species of
Characteristic | HN31(22) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
---|---|---|---|---|---|---|---|---|---|---|---|
Morphology | Short roads | Short roads | Short roads | Short roads | Short roads | Cocci | Short roads | Cocci | Cocci | Cocci | Cocci |
Tincion | HN31(22) | Gram + | Gram + | Gram + | Gram + | Gram + | Gram + | Gram + | Gram + | Gram + | Gram + |
Colony pigmentation | Yellow | Yellow | Yellow | Yellow | Yellow | Orange/Yellow | Yellow | Orange/Yellow | Yellow | Yellow | White ivory |
Temperature (°C) | 30–40 | 40–42 | 8.5–42 | 35–37 | 20–40 | 4–40 | 25–40 | 4–36 | 4–36 | 4–36 | NR |
pH | 5–11 | 8–12 | 7.5–9.5 | 7–11 | 7–12 | 7–9 | 7–11 | 5–12 | 6.5–10 | 6–8.5 | 6–10.5 |
NaCl (%) | 0–20 | 0–10 | 0–15 | 0–18 | 0–25 | 0–25 | 3–12 | 1–15 | 0–20 | 0–16 | 0.5–30 |
Catalase | + | + | NR | + | NR | NR | + | NR | NR | NR | NR |
Oxidase | – | – | – | – | – | – | + | – | – | – | – |
Urease | – | NR | NR | NR | + | + | – | – | – | – | NR |
Nitrate reduction | – | – | – | + | – | – | NR | – | + | – | + |
Citrate test | + | – | W | – | NR | NR | – | NR | NR | NR | NR |
Voges-Proskauer | – | – | – | – | NR | NR | – | – | – | + | NR |
Acid produced from: | |||||||||||
Galactose | – | – | – | – | – | – | – | + | + | + | NR |
Trehalose | + | – | + | – | – | – | – | W | + | W | – |
Mannitol | + | – | – | – | – | – | – | + | + | + | NR |
Sucrose | + | NR | NR | – | – | – | – | NR | NR | NR | – |
Fructose | + | NR | NR | + | – | – | + | NR | NR | NR | NR |
Utilization of: | |||||||||||
Glucose | + | + | NR | + | + | + | + | + | W | + | + |
Trehalose | + | – | + | – | – | – | – | + | – | + | NR |
Fructose | – | + | + | + | + | + | + | + | + | + | + |
Mannose | + | + | + | + | + | + | – | + | + | W | + |
Sucrose | + | + | NR | + | + | + | W | + | + | + | NR |
Maltose | + | + | NR | – | + | + | NR | + | + | + | NR |
Hydrolysis of: | |||||||||||
Starch | + | + | – | + | – | – | + | – | + | – | – |
Gelatin | + | + | – | NR | + | + | + | + | – | – | NR |
Tween 80 | + | + | – | + | NR | NR | NR | – | – | – | – |
Casein | + | NR | NR | – | + | – | + | – | + | NR | – |
(+) Positive, (–) negative, (w) weak reaction, (NR) unreported.
Research studies performed on the microorganisms of the Texcoco Lake, showed that there has been a great microbial adaptation to the extreme environmental conditions (Luna-Guido and Dendooven 2001; Soto-Padilla et al. 2014; Valenzuela-Encinas et al. 2008). Soto-Padilla et al. (2014) reported the presence of the genera with halophile and alkaline characteristics, such as:
The