Diesel degradation in soil catalyzed by the addition of a bioagent

Abstract

The supernatant harvested from a mesophilic, molasses-fed, non-methanogenic bioreactor, which is rich in nitrogen, phosphorous, metals, free amino acids, Sporolactobacillus sp., Prevotella sp., and Clostridium sp., is diluted with tap water and tested as the bioagent to catalyze diesel degradation in soil. Outdoor experiments are performed under the following conditions to assess the effectiveness of the bioagent: diesel doses: 9–15 mg TPH/g soil; bioagent concentrations and dose: 1–5 % at 60 ml/day; soil sample size: 600 g; ambient temperatures: 28–32 °C, and relative humidity: 40–82 % (TPH: total petroleum hydrocarbons). Diesel degradation in soil treated with 3 % bioagent, which proceeds at the rates of 1.04–1.55 mg TPH/g soil-day, is completed in about a week with up to 83 % efficiencies. In contrast, diesel degradation in soil sprinkled with water (60 ml/day) proceeds at the rate of 0.3 mg TPH/g soil-day that achieves 15–22 % degradation efficiencies. The addition of 3 % bioagent yields desired soil moisture content (10–15 %), soil pH (6.8–8.2), and nutrition inputs. Both Sporolactobacillus sp. and Prevotella sp. grown on molasses are robust, tolerant high diesel doses, able to utilize hydrophobic hydrocarbons, and readily adaptable to the soil environment. Most notably, prior acclimatization is not required to enable these properties.

Appendix 1: PCR and RT-PCR procedures (Chang et al. 2008; Chou et al. 2011)

For PCR tests, DNA was extracted using FastDNA™ Spin Kit for Soil (MP Biomedicals, USA). The samples were processed following the procedures specified in the vendor manual. Table 3 list the primers sets used. Each reaction was performed with a mixture of 3 μl of template DNA, 2 μl (10 μM) of each primer set, 33 μl of double-distilled water and 10 μl of PCR Master Mix (BioKit, Taiwan). The thermal cycling program was: 2 min at 95 °C, 30 30-s cycles at 95 °C, 30 s at 54 °C for Prevo F/R and Sporo F/R (50 °C for the Clos L1F/R and Clos E1F/R), 30 s at 72 °C, and the program was terminated at 72 °C for 10 min. PCR results were checked with gel electrophoresis.

Table 3 Primer sets

The fluorescent dye, SsoFast™ EvaGreen^® Supermix, was used for real-time PCR (RT-PCR) reactions using the procedure specified in the vendor’s manual (Bio-Rad, Hercules, USA). Reactions were performed in a 48-well optical reaction plate and set into a 48-well MJ Mini™ Gradient Thermal-Cycler (Bio-Rad, Hercules, USA). Primers sets are listed in Table 3. Each reaction was performed in triplicate with a mixture of 2 μl of cDNA, 1 μl (3–5 μM) of each primer set, 2 μl of double-distilled water and 5 μl of enzyme Supermix. The thermal cycling program was: 60 s at 95 °C, 40 5-second cycles of at 95 °C, 5 s at 54 °C for Prevo F/R and Sporo F/R (or 50 °C for Clos L1F/R and Clos E1F/R) and followed by a dissociation stage (i.e., 15 s at 95 °C, 15 s at 65 °C, and followed by a slow temperature rise to 95 °C). Neither primer-dimer artifacts nor nonspecific PCR amplicons were observed during the melting process. The efficiency of each reaction was between 90 % and 110 %. R² values for all standard curves were greater than 0.99.