| Execution Methods |
- Sampling and cultivation of bacterial cultures: Anaerobic reduction and dehalogenation colonies are collected from a TCE-contaminated site in southern Taiwan. The sampling depth is determined according to the distribution of contaminants and the measurement results of dissolved oxygen (or redox potential). The sampling location of anaerobic bacteria is better to be close to the source of pollution. Usually, the main terminal electron accepting processes at this location have reached the stage of methanation or halo-respiration. Thus, it should be possible to obtain local bacteria with higher decomposition ability to TCE. Anaerobic bacteria were partially acclimated by adding1.0 mg/L of TCE.
- Taguchi biodegradation experiment: A total of 10 groups of Taguchi experiments with 4 control factors were conducted (1 group of blank + 9 groups of Taguchi experimental design according to the L9(34) orthogonal table). Using 50-95°C to select 3 different temperatures, and the exposure time of 45 minutes is the control factor A. The pH value of the heat selection is the control factor B. The salinity of the heat selection is the control factor C and the moisture content is the control factor D. Taguchi experiment can help finding the optimum selection conditions. This part will produce at least 90 groups of samples for 16S rRNA gene sequencing.
- Metagenome analysis: This includes DNA extraction, Real-time Polymerase Chain Reaction (referred to as RT-PCR or qPCR) and 16S rRNA gene sequencing analysis.
- Re-spike experiment: By selecting the two groups with top performance for a re-spike experiment. Each addition was aimed to reach the concentration of 10.0 mg/L as the target. Three experiments were conducted to determine the highest rate of TCE degradation by the selected group.
- Publication of results: 1 patent, 2 SCI journal papers and at least 2 seminar papers will be produced.
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| Performance Evaluation |
1. The specific results of this plan are as follows: - Completed the full-length sequencing of the third-generation 16S rRNA gene in 2 monitoring wells in the trichloroethylene (TCE)-contaminated site, TCE decomposing culture sampling, and 10 groups of samples in the Taguchi test. Completed 180 sets of 16S rRNA gene data sets and it was found that the microbiomes have been reconstructed trough the selection process.
- The optimal screening conditions are defined as 50ºC, pH 9, 100% seawater salinity and 21% volumetric water content (θW), and the conditions are repeatable indicating that this method has high engineering feasibility.
- The best group of bacterial community containing only 1.23% Dehalococcoides mccartyi 192 can completely dechlorinate 11.81 mg/L TCE into ethene within 3.0 days, which is about 2.5-100 times faster than the rates reported in international literature. It is the fastest rate in the world at present, and has the opportunity to open up new opportunities for soil and groundwater biological remediation related industries in Taiwan.
- The interaction between Dehalococcoides mccartyi 192 and Desulfuromonas michiganensis and Cloacibacillus porcorum was found, which has never been reported in the current literature.
- Redundancy analysis confirmed that four environmental factors can effectively reshape microbiomes, and temperature and salinity are the main and mututally independent factors, among which temperature is the most important factor causing the largest difference in microbiome structure.
2. Application to related programs: At present, this lab has received NT$4,680,000 of government subsidies and industry-university cooperation programs and continues to develop related technologies, as described below. - Special project of the Ministry of Science and Technology: MOST 109-2221-E-005 -005 Research on local high-efficiency dioxin pollutant-degrading bacteria using microbiome reengineering (MBRE) technology, NT$1,390,000 (2021.08.01-2022.07.31) .
- Environmental Protection Agency Soil Foundation Project: Erren River Contaminated Sediment Remediation Pilot Study Project (polybrominated diphenyl ether and polycyclic aromatic hydrocarbon pollution remediation technology research and development), NT$2,610,000 (2021.02.24-2022.02. 28).
- Industry-university cooperation plan: "The service plan for the preparation, perfusion, decomposing bacteria purification and seed identification of chlorinated ethylene pollution control aids" by Shuichuang Technology (stock) Company, NT$$680,000 (2021.08.01-2022.01. 31).
- It has been confirmed that we will participate in the EPA Soil Foundation Soil and Groundwater Microbial Gene Database Platform Project.
- In December 2021, we have applied to the Ministry of Science and Technology a 2-year project for the purification of TCE rapid degrading bacteria and research on special functional genes.
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| Conclusion & Suggestion |
- This project reveals that the biological remediation of environmental groundwater TCE pollution can actually be achieved by selecting the local microbial flora, without the need to inject foreign bacteria, which is the best solution to reduce biosafety concerns and lateral transfer of genes. .
- Due to the excellent implementation results, a large domestic petrochemical group has negotiated with me to conduct a similar experiment due to its own TCE-contaminated groundwater remediation problem, hoping to select the right microorganisms for its contaminated site.
- Due to the excellent project results, another domestic environmental engineering consulting firm has decided to cooperate with my laboratory to conduct the pilot study of its remediation site, and will also use this MBRE technology to conduct a pilot study.
- In March 2022, the Taiwan-Vietnam International Symposium hosted by my laboratory will be published, and the Vietnamese side will be invited to carry out the continuous development and application of this technology. The international cooperation part will be jointly carried out by domestic engineering consulting companies.
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