Antarctic bacteria as bioremediation tools

RUBERTO L; VAZQUEZ SC; DIAS R; MAC CORMACK WP

Rosario, ARG

Taller; 3er Taller de Criobiología Aplicada a las Ciencias Médicas; 2009

UNR - CBCCA Unesco Chair in Criobiology

Introduction Bioremediation of cold soils require the use of low-temperature adapted organisms (Aislabie et al, 2006). The most adequate places to obtain cold-adapted microorganisms are those permanently exposed to low temperature. In this sense, Antarctic soils are considered as model habitats to searching for this kind of microorganisms. Cold-adapted organisms are generally assorted into two groups: psychophiles and psychrothrops (or psychrotolerant). Psychrophilic bacteria are able to growth optimally below 15°C but are also unable to grow above 20°C. Psychrotolerant bacteria are able to grow at low temperatures but are not restricted to such condition and have optimum growth levels above 20°C (Morita, 1975). In summer, temperature in some regions of the Antarctic Peninsula can reach 10°C during sunny days rising soil temperature to 15 -20°C. For this reason, psychrotolerant bacteria represent the most adequate option for using in any soil bioremediation processes under these conditions. The aim of this work was the isolation and characterization of psychrotolerant bacterial strains and consortia and the evaluation of their hydrocarbon removal efficiency under field assays carried out in Antarctica. Isolation of the bacterial tools The bacterial strains and consortia were isolated by enrichment cultures with hydrocarbons as sole carbon source (Jobson et al, 1972). In this sense, strain DM1-41 was obtained using crude oil. Strains MP2-4 and M10dp and consortia J13 and M10 were obtained using polycyclic aromatic hydrocarbons (PAHs). All the strains were psychrotolerant gram negative rods. The partial sequence of the 16S DNAr showed that DM1-41 is related with Stenotrophomonas rhizophila (T) (99,7%), MP2-4 with Pseudomonas migulae (T) (99,4%) and M10dp with Sphingobium xenophagum (T) (97,3%). All these genera are referred as common inhabitants of Antarctic hydrocarbon contaminated soils (Stallwood, 2005; Saul et al, 2005). In order to isolate the main components of M10, an analysis of the culturable components of the consortia was also performed.. Having these M10-derived isolates it would be possible to reconstruct the consortia in a reproducible and defined way for their use as innoculum. With this objective in mind, two liquid cultures of M10 were carried out using either gasoil (GO) or phenanthrene (Phe) as sole carbon and energy source. After 24 and 192 h cultures were sampled and serial dilution were plated in agarized saline basal media (SBM) containing gasoil or a mix of GO and Phe as carbon source. Four sets of isolates were obtained. Using RISA (ribosomal intergenic spacer analysis) the isolates were grouped in clusters and representative members of each group were identified by partial sequencing of 16s DNAr. Three main groups were detected: Pseudomonas, Stenothrophomonas and Pedobacter.However, members of the Pseudomonadaceae family were by far the most abundant ones. When the growth capacity of M10 consortia at different temperatures was analyzed, the results suggested that it is a psychrotolerant consortium rather than a psycrophilic one. Using GO as substrate, apparent growth rate at 20, 15 and 10°C were 0,350 h-1; 0,078 h-1 and 0,032 h-1 respectively. Stenotrophomonas rhizophila (T) (99,7%), MP2-4 with Pseudomonas migulae (T) (99,4%) and M10dp with Sphingobium xenophagum (T) (97,3%). All these genera are referred as common inhabitants of Antarctic hydrocarbon contaminated soils (Stallwood, 2005; Saul et al, 2005). In order to isolate the main components of M10, an analysis of the culturable components of the consortia was also performed.. Having these M10-derived isolates it would be possible to reconstruct the consortia in a reproducible and defined way for their use as innoculum. With this objective in mind, two liquid cultures of M10 were carried out using either gasoil (GO) or phenanthrene (Phe) as sole carbon and energy source. After 24 and 192 h cultures were sampled and serial dilution were plated in agarized saline basal media (SBM) containing gasoil or a mix of GO and Phe as carbon source. Four sets of isolates were obtained. Using RISA (ribosomal intergenic spacer analysis) the isolates were grouped in clusters and representative members of each group were identified by partial sequencing of 16s DNAr. Three main groups were detected: Pseudomonas, Stenothrophomonas and Pedobacter.However, members of the Pseudomonadaceae family were by far the most abundant ones. When the growth capacity of M10 consortia at different temperatures was analyzed, the results suggested that it is a psychrotolerant consortium rather than a psycrophilic one. Using GO as substrate, apparent growth rate at 20, 15 and 10°C were 0,350 h-1; 0,078 h-1 and 0,032 h-1 respectively. Pseudomonas, Stenothrophomonas and Pedobacter.However, members of the Pseudomonadaceae family were by far the most abundant ones. When the growth capacity of M10 consortia at different temperatures was analyzed, the results suggested that it is a psychrotolerant consortium rather than a psycrophilic one. Using GO as substrate, apparent growth rate at 20, 15 and 10°C were 0,350 h-1; 0,078 h-1 and 0,032 h-1 respectively. -1; 0,078 h-1 and 0,032 h-1 respectively. On site soil bioremediation assay in Antarctica A bioremediation field assay was made using chronically hydrocarbon contaminated soil from Marambio Station. The assay was carried out in microcosms performed in metal trays containing 2,5 kg of soil. The experiment includes: abiotic control (AC), community control (CC), biostimulation of the authocthonous microflora (BAM), and three systems where biostimulation was combined with bioaugmentation: i) mix of bacterial strains described previously (S+B); ii) M10 consortium (M10+B); J13 consortium (J13+B). During the assay, temperature range was -7.8°C-+7.3°C (mean: + 0.5°C) and included 26 days in which temperature dropped below 0°C. Counts of total heterotrophic aerobic bacteria (THAB) and hydrocarbon degrading bacteria (HDB) were analyzed by plate-counting using caseine-peptone-starch (CPS) agar and agarized MSB-GO (AGO) respectively. Total petroleum hydrocarbons (TPH) were quantified by FT-IR (EPA 418 method). Results showed a rise in biological activity (figure 1) evidenced as an increase in THAB and HDB counts. At the end of the assay (45 d) the number of HDB was significantly higher in all systems (BAM, M10+B, S+B and J13+B) compared with the control system. TPH decreased from the initial concentration (11.972 ppm) to 6.460 ppm in the abiotic control (AC) and 1.687 ppm in the M10+B system. However, the efficiency of hydrocarbon removal (figure 2) observed in M10+B was not significantly different from those obtained with the BAM. Conclusions soil bioremediation assay in Antarctica A bioremediation field assay was made using chronically hydrocarbon contaminated soil from Marambio Station. The assay was carried out in microcosms performed in metal trays containing 2,5 kg of soil. The experiment includes: abiotic control (AC), community control (CC), biostimulation of the authocthonous microflora (BAM), and three systems where biostimulation was combined with bioaugmentation: i) mix of bacterial strains described previously (S+B); ii) M10 consortium (M10+B); J13 consortium (J13+B). During the assay, temperature range was -7.8°C-+7.3°C (mean: + 0.5°C) and included 26 days in which temperature dropped below 0°C. Counts of total heterotrophic aerobic bacteria (THAB) and hydrocarbon degrading bacteria (HDB) were analyzed by plate-counting using caseine-peptone-starch (CPS) agar and agarized MSB-GO (AGO) respectively. Total petroleum hydrocarbons (TPH) were quantified by FT-IR (EPA 418 method). Results showed a rise in biological activity (figure 1) evidenced as an increase in THAB and HDB counts. At the end of the assay (45 d) the number of HDB was significantly higher in all systems (BAM, M10+B, S+B and J13+B) compared with the control system. TPH decreased from the initial concentration (11.972 ppm) to 6.460 ppm in the abiotic control (AC) and 1.687 ppm in the M10+B system. However, the efficiency of hydrocarbon removal (figure 2) observed in M10+B was not significantly different from those obtained with the BAM. Conclusions On site bioremediation of contaminated cold environments, like Antarctic soils, is possible using the potential of psychrotolerant bacteria and the biostimulation strategy. Bioaugmentation combined with biostimulation seemed to be not advantageous compared with biostimulation only. Figure 1. Evolution of THAB and HDB counts during the field assay. Figure 2. TPH at day 1 and day 45 and removal efficiency (%) for the different systems. References Aislabie J, Saul D, Foght J. (2006). Bioremediation of hydrocarbon contaminated polar soils. Extremophiles. 10:171-179. Jobson A, Cook D, Westlake D. (1972). Microbial utilization of crude oil. Appl Microbiol. 23:1082-1089. Morita RY. (1975) Psychrophilic bacteria. Bact Rev. 39:144-167. Saul D, Aislabie J, Brown C, Harris L, Foght J. (2005). Hydrocarbon contamination changes the bacterial diversity of soil from around Scott Base, Antarctica. FEMS Microbiol Ecol. 53: 141-155. Stallwood B, Shears J, Williams P, Hughes K. (2005). Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica. J Appl Microbiol. 99: 794-802 bioremediation of contaminated cold environments, like Antarctic soils, is possible using the potential of psychrotolerant bacteria and the biostimulation strategy. Bioaugmentation combined with biostimulation seemed to be not advantageous compared with biostimulation only. Figure 1. Evolution of THAB and HDB counts during the field assay. Figure 2. TPH at day 1 and day 45 and removal efficiency (%) for the different systems. References Aislabie J, Saul D, Foght J. (2006). Bioremediation of hydrocarbon contaminated polar soils. Extremophiles. 10:171-179. Jobson A, Cook D, Westlake D. (1972). Microbial utilization of crude oil. Appl Microbiol. 23:1082-1089. Morita RY. (1975) Psychrophilic bacteria. Bact Rev. 39:144-167. Saul D, Aislabie J, Brown C, Harris L, Foght J. (2005). Hydrocarbon contamination changes the bacterial diversity of soil from around Scott Base, Antarctica. FEMS Microbiol Ecol. 53: 141-155. Stallwood B, Shears J, Williams P, Hughes K. (2005). Low temperature bioremediation of oil-contaminated soil using biostimulation and bioaugmentation with a Pseudomonas sp. from maritime Antarctica. J Appl Microbiol. 99: 794-802 Pseudomonas sp. from maritime Antarctica. J Appl Microbiol. 99: 794-802