Comparison on the measurement method of soil live bacterial number as part of the biogeochemical site characteristics

Jeonggil Lee 고려대학교 그린스쿨대학원 2020 국내석사

Soils are rich in bacteria (1 × 10 2–2 × 109 cells g-1), with live bacterial abundance dependent on a number of physical and chemical properties, such as organic matter, temperature, particle distribution, moisture, and pH. Soil bacteria play a significant role in global nutrient cycling as well as soil biogeochemistry. There are a number of different methods that have been used to quantify live bacteria in soil. To date, an evaluation of these methods has not been undertaken to identify the most precise and accurate method(s). This study evaluates the conditions of appropriate storage temperature conditions (i.e., -20, 4, 24, 30 °C) and pretreatment methods (i.e., sonication, centrifugation, filtration) for measuring live soil bacterial populations. We also investigated culture-dependent methods (CDMs; i.e., colony forming unit (CFU), spotting, and most probable number (MPN)) and culture-independent/direct counting methods (CIMs; i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA extraction). Each method was tested using 72 soil samples collected from a local farm site at three different depths (i.e., 20, 100, and 180 cm). As the storage temperature at 4 °C showed the lowest variation of live bacterial cell number, storage at 4 °C was most appropriate, and the highest number of live bacterial populations was measured with the pretreatment of 3 minutes of sonication time (300 W), 1400 × g of centrifugation speed. Among all CDMs, MPN was found to be rapid, simple, and reliable (low variability amongst triplet). However, the number of bacteria quantified by MPN was 1–2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA extraction method appeared to overestimate soil bacterial numbers, which may be attributed to DNA extraction from dead bacteria and free DNA in the soil - VIII matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Bacterial numbers obtained using CDMs and CIMs were also compared based on soil pH, soil water content, and clay/silt content. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead), and their spatial link to soil physical and chemical properties.

Comparison on the measurement method of soil live bacterial number as part of the biogeochemical site characteristics

이정길 Green School Graduate School of Energy and Environ 2020 국내석사

Distribution and geochemical characteristics of Sb and toxic metals near an antimony refinery and their impact on indigenous microbes

박수찬 Graduate School, Korea University 2020 국내석사

Although several studies have investigated the effects of Sb contamination on surrounding environments and indigenous microorganisms, little is known about the effect of co-contamination of Sb and toxic metals. In this study, the occurrence of Sb and other toxic metals (e.g., As, Pb) near an operating Sb refinery and near-field landfill site were investigated. Topsoil samples near the refinery show high levels of Sb (~3,250 mg kg-1) but relatively low concentrations of other toxic metals. However, several deeper soil samples from the near-field landfill site contain high concentrations of As and Pb as well as extremely high Sb contents (~21,400 mg kg-1). X-ray absorption fine structure analysis shows that Sb in soils from both sites is present as Sb(V) in the form of tripuhyite (FeSbO4), a stable mineral phase. Three-dimensional principal coordinates analysis results show that microbial community compositions in samples with high toxic metal concentrations are significantly different from other samples and have lower microbial populations (~104 MPN g-1). Sequential extraction results reveal that Sb is present primarily in the residual fraction (~99 %), suggesting low Sb bioavailability. However, redundancy analysis suggests that easily extractable Pb might be the major factor controlling microbial community compositions at the site.

Biogeochemical characteristics of seepage water and mineral precipitates in the underground silo disposal facility

함박눈 Green School Graduate School of Energy and Environ 2020 국내박사

Constructing an underground silo disposal facility can cause dramatic changes in groundwater quality, flow direction, and level, which may also influence subsurface microbial activity and community composition. We collected and characterized multiple samples within an underground disposal facility near the east coast in South Korea, including seepage water samples downgradient from the silos and tunnels, as well as precipitates deposited along the flow path of the seepage water. To understand how the construction of the underground structures influenced the subsurface environment, we investigated seepage water chemistry, mineral precipitates’ characteristics as determined by X-ray analysis (XRD and XAFS), and microbial community composition determined by 16S rRNA gene analysis at several different locations in a disposal facility. Some seepage waters showed Ca-Cl type with relatively high TDS, while others were Ca-Na-HCO3 type. The ratios of Cl and δ18O showed that the Ca-Cl type seepage waters were influenced by seawater at the varying extent of 2.7─15.1%. Various sulfate-reducing bacteria were identified in Ca-Cl type seepage waters with relatively high sulfate content due to seawater intrusion. Some samples showed an extremely high pH (>10) and a very high abundance of Hydrogenophaga. The precipitates deposited along the flow path of the seepage water showed a wide range of mineral phases including calcite, ferrihydrite, green rust, and siderite depending on the seepage water chemistry and microbial activity. This study suggests that the construction of underground disposal facility structures can create distinct, localized geochemical conditions (i.e., high alkalinity, salinity, and oxic condition) and impact microbial community structure. The changes in geochemical conditions may stimulate biological sulfate reduction and also result in pump clogging by mineral precipitation. Long-term monitoring is essential to better predict and assess the safety of this disposal facility and other underground structures.

Microbial Reactions by Indigenous Microbes in Bentonite under Deep Geological Repository Environments

Kanghyun Park 고려대학교 대학원 2025 국내박사

Reduction and Transformation of Antimony(V) Phases Driven by Microbiological and Geochemical Processes in Subsurface Environments

ZHANG YIDAN 고려대학교 대학원 2025 국내박사

Antimony (Sb), a group 15 chalcophile metalloid, naturally occurs through hydrothermal processes and the weathering of Sb-bearing minerals. However, elevated Sb concentrations are often introduced into the environment due to anthropogenic activities such as mining, and smelting and refining of Sb ores. Another source of Sb release is spent bullets deposited in soils and sediments at shooting ranges, where Sb is used as a hardening agent in ammunitions. Sb is a non-essential element, and human exposure to it can lead to various health hazards, including respiratory diseases, neural damage, and potentially cancer. Sb commonly exists in two oxidation states: Sb(III) and Sb(V). While Sb(III) is more toxic, its tendency to form insoluble phases can immobilize it, reducing its ecotoxicity. In oxic environments, Sb(V) has been found to be the dominant species, often present as a range of Sb(V)-bearing solid phases. Nonetheless, Sb(V) can be more mobile, particularly in anoxic environments or in association with organic matters. Changes in redox conditions can facilitate the reduction of Sb(V) to Sb(III), a process of concern due to the increased toxicity of Sb(III). However, Sb(V) reduction could also be explored as a remediation strategy by immobilizing Sb(III) in solid phases. Therefore, investigating Sb(V) reduction is crucial for advancing the understanding of Sb natural cycling and for developing effective strategies to manage Sb contamination. This thesis is therefore centered around Sb(V) reduction, with each chapter addressing specific questions that explore the factors controlling Sb(V) reduction, the mechanisms underlying these processes, and the broader environmental significance of the results. The aim is to integrate the findings from each chapter to contribute to a comprehensive understanding of Sb cycling, and ultimately support the goal of achieving efficient Sb management. Chapter 1 provides a literature review on the current research progress and understanding of Sb(V) reduction, which can be classified into abiotic, biotic, and coupled biotic-abiotic processes. This chapter systematically summarizes the key studies on these topics, highlighting their major findings. This review not only builds essential background knowledge but also identifies knowledge gaps, which the experiments in this thesis are designed to address. Chapter 2 presents a field study in which soil and rock samples were collected from three Sb-contaminated sites to compare the biogeochemical factors influenced by Sb presence. To understand Sb behavior in the environment, it is necessary to first identify the forms in which Sb occurs upon release from contamination sources. This provides insight into the potential biogeochemical processes affecting Sb. Additionally, assessing the correlation between Sb and local biogeochemical parameters at contaminated sites enhances the understanding of Sb as an environmental contaminant. Chapter 3 documents an isolation study aimed at acquiring a novel strain of Sb(V)-reducing bacterium using Sb-contaminated soil as the source. Microbial Sb(V) reduction holds promise as an effective and eco-friendly Sb remediation strategy, though many aspects of this process remain unclear. Isolating a model Sb(V)-reducing strain allows the investigation of the reduction mechanisms, the formation of Sb(III) biominerals, and the molecular pathways involved in this process. Chapter 4 details a microcosm study that investigated the fate of Sb(V) associated with ferrihydrite under potential competition with the oxyanion phosphate (PO43-) and in iron(Fe(III))- and sulfate-reducing conditions. The immobilization of Sb(V) in anoxic environments can be achieved by its adsorption onto ferric solid phases, such as ferrihydrite. However, the adsorption of the Sb(V) oxyanion, Sb(OH)6-, may be challenged by competing anions such as PO43-. Furthermore, the transformation of the host ferric phase by Fe(II) and sulfide produced during microbial activities can significantly impact Sb mobility and speciation. This chapter thoroughly examined the competitive adsorption between Sb(V) and PO43- with ferrihydrite as the host phase, and explores whether such competition affects Sb(V) behavior during reductive dissolution of ferrihydrite stimulated by microbial Fe(III) and sulfate reduction. Chapter 5 is a combined microcosm and flow-through column study aimed at exploring the transformation of tripuhyite induced by aqueous Fe(II). Tripuhyite (FeSbO4) is a crystalline phase commonly found at many Sb-contaminated sites and is considered as a sink for Sb(V). However, the alteration in Sb mobility and speciation during reductive dissolution of FeSbO4 is unknown. This chapter probed the potential transformation of FeSbO4 through biogenic Fe(II) produced by microorganisms and abiotic Fe(II) introduced via flow-through. The column setup also accounts for realistic transportation processes in the environment. This dual approach provides a comprehensive framework to investigate the fate of Sb during transformation of the crystalline host phases. 안티모니(Sb)는 15족 친유황 준금속으로, 자연적으로는 Sb 함유 광물의 열수작용 및 풍화 과정을 통해 생성된다. 그러나 Sb의 농도가 높아지는 주요 원인은 광산 채굴, 제련 및 정제와 같은 인간 활동에 기인한다. 또 다른 Sb의 주요 방출원은 사격장에서 토양과 퇴적물에 축적된 소모된 탄환으로, Sb는 탄약의 경화제로 사용된다. Sb는 필수적인 원소가 아니며, 인간이 Sb에 노출될 경우 호흡기 질환, 신경 손상 및 잠재적으로 암과 같은 다양한 건강 문제를 초래할 수 있다. Sb는 일반적으로 두 가지 산화 상태인 Sb(III)와 Sb(V)로 존재한다. Sb(III)는 독성이 더 강하지만 불용성 상을 형성하는 경향이 있어 이동성이 줄어들고 생태 독성이 감소할 수 있다. 반면에, 산소가 풍부한 환경에서는 Sb(V)가 주요 종으로 존재하며, 종종 Sb(V)-함유 고체 상으로 나타난다. 그러나 Sb(V)는 특히 무산소 환경이나 유기 물질과 결합할 때 더 이동성이 높아질 수 있다. 산화환원 조건의 변화는 Sb(V)가 Sb(III)로 환원되는 과정을 촉진할 수 있는데, 이는 Sb(III)의 독성이 증가하기 때문에 중요한 우려 사항이다. 하지만 Sb(III)를 고체 상으로 고정화함으로써 Sb(V) 환원을 정화 전략으로 활용할 수도 있다. 따라서 Sb(V) 환원을 연구하는 것은 Sb의 자연 순환에 대한 이해를 심화시키고 Sb 오염을 관리하기 위한 효과적인 전략을 개발하는 데 매우 중요하다. 이 논문은 Sb(V) 환원에 초점을 맞추고 있으며, 각 장은 Sb(V) 환원을 제어하는 요인, 이러한 과정의 메커니즘, 그리고 결과의 더 넓은 환경적 의미를 탐구하는 특정 질문을 다루고 있다. 각 장의 결과를 통합하여 Sb 순환에 대한 포괄적인 이해를 제공하고, 궁극적으로 효과적인 Sb 관리 목표를 달성하는 데 기여한다.

Microbial Fe(III) reduction across a pH gradient: The impacts on secondary mineralization and microbial community development

Yun Seo Jang 고려대학교 대학원 2025 국내석사

Fe(III) oxide-hydroxides are prevalent in natural environments and play a key role in contaminants or nutrients cycling. In anoxic environments, Fe(III) reduction is largely facilitated by dissimilatory Fe(III) reducing bacteria (DIRB). Dissimilatory iron reduction (DIR) results in the reductive dissolution of Fe(III) phases and subsequent secondary mineralization. These processes are strongly influenced by changes in pH, as protons serve as reactants in DIR, and pH levels impact the extent of Fe transformation. Currently, there is a lack of studies investigating DIR under environmentally relevant pH gradients and its impact on secondary mineralization and microbial community development. This study aimed to understand the impact of an initial pH gradient (6.3, 6.9, 7.3, 7.7, and 9) on DIR, using acetate as the electron donor, and goethite or lepidocrocite as the electron acceptor. Lepidocrocite, with its relatively lower crystallinity compared to goethite, allowed for greater DIR. However, the rate and extent of Fe(III) reduction consistently decreased as pH increased from 6.3 to 9. Solid phase analyses revealed predominant siderite formation at pH 6.3 and 6.9 following lepidocrocite reduction, with partial transformation to siderite at pH 7.3 and 7.7. This was due to the positively charged Fe(III) surface preventing aqueous Fe(II) adsorption, leading to its precipitation with bicarbonate from the medium. Minor formation of goethite and magnetite was also observed in lepidocrocite-amended microcosms at pH 6.3 and 6.9, resulting from extensive Fe(II) production. Conversely, more stable goethite showed no transformation, despite the observed Fe(II) production. 16S rRNA sequencing indicated greater enrichment of DIRB at lower pH, but a decline in abundance as pH increased. Overall, these findings demonstrate that pH exerts a more substantial influence on DIR than other factors, such as Fe mineralogy. This study highlights pH as a critical factor directly impacting DIR, secondary mineralization, and DIRB development. 철(III)수산화광물은 자연계에 널리 분포해 있고 오염물질과 영양분 순환에 중요한 역할을 한다. 혐기성 환경에서 철(III) 환원은 철환원박테리아(DIRB)에 의해 이루어지고, 철(III) 환원반응 (DIR) 으로 환원성 용해와 2차광물 침전이 일어날 수 있다. 수소이온이 DIR에 반응물로 소모되고 pH 의 단위에 따라 철광물의 변화 정도에 다르기 때문에, DIR 은 pH 의 변화에 크게 영향을 받을 수 있다. 자연환경에 적합한 pH에서의 철(III) 환원 (DIR) 과 2차광물 생성 및 미생물 군집에 대한 연구는 현재 부족하기 때문에 본 연구는 초기 pH (6.3, 6.9, 7.3, 7.7, 9)가 DIR에 주는 영향을 규명하고자, acetate와 goethite/lepidocrocite 를 각각 전자공여체와 전자수용체로한 microcosm 실험을 진행하였다. Goethite 보다 낮은 결정성을 가진 lepidocrocite 실험에서 철(III) 환원 정도가 더 높았지만 두가지 철(III) 광물 모두 pH가 올라갈수록 환원 정도가 감소하였다. 고체상 분석 결과, lepidocrocite에서 철(III)의 환원으로 pH 6.3 과 pH 6.9에서 siderite 가 우세하게 형성되었고, pH 7.3과 pH 7.7에서는 소량으로 형성되었다. Siderite의 형성은 산성환경에서 양성전하로 된 철(III) 광물의 표면이 용존 철(II) 흡착을 억제하여, 용존 철(II) 과 배지상 bicarbonate의 침전으로 기인한 것으로 사료된다. Lepidocrocite실험의 pH 6.3과 pH 6.9에서 철(III) 환원 반응으로 소량의 goethite와 magnetite도 형성되었다. 반면, goethite 실험에서는 철(II) 가 생성되었지만, goethite 의 결정성이 높은 성질로 광물의 변화가 일어나지 않았다. 16S rRNA 분석결과, 낮은 pH 에서 높은 비중의 철환원미생물(DIRB)과 높은 pH 일수록 낮은 비중의 DIRB를 나타내었고, 전반적으로 pH 가 철 광물의 종류 보다 철(III)환원 (DIR) 반응에 더 상당한 영향을 준다는 것을 시사한다. 본 연구는 DIR, 2차 광물 생성, 미생물 군집 진화에 pH 가 주요인임을 강조한다.

Temperature-dependent microbial reactions by indigenous microbes in WRK bentonil under Fe(III)- and sulfate- reducing conditions

Park, Su Young 고려대학교 대학원 2023 국내석사

벤토나이트는 높은 팽윤능력과 같은 지질공학적 특성으로 인해 사용후핵연료의 심부 지중 처분에서 공학적 방벽 구성물질로써 가장 적합한 물질로 여겨지고 있다. 사용후핵연료는 일반적으로 금속보관재 (구리, 탄소강)에 밀폐되어 결정질 암반에 저장되게 되고, 벤토나이트는 방사성 핵종 누출시 이를 저지하기 위해 금속보관재와 결정질 암반 사이의 채움재로써 사용되게 된다. 하지만, 처분장 건설시 벤토나이트에 존재하는 토착미생물 또한 심부 지중환경으로 유입될 수 있다. 이렇게 유입된 벤토나이트의 토착미생물은 다양한 미생물학적 반응들을 통해 저장소의 안정성에 영향을 줄 수 있다. 또한, 금속보관재 주변은 방사성 핵종의 붕괴열로 인해 고온환경이 형성될 수 있다. 따라서 본 연구는 WRK 벤토나이트에 존재하는 토착미생물의 존재를 확인하고 온도 조건이 미생물 활동에 미치는 영향을 조사하였다. 일반적인 지하수 온도 (18℃)와 고온 (50℃) 조건에서 약 9개월간 배치 실험을 진행하였고, 전자공여체로는 락테이트, 전자수용체로는 페리하이드라이트 그리고/또는 황산염을 사용하였다. 실험 초기에 락테이트는 18℃ 조건에서는 아세테이트와 프로피오네이트로 분해되었지만, 50℃ 조건에서는 주로 아세테이트로 분해되었다. 철환원의 정도는 50℃ 보다 18℃ 조건에서 크게 나타난 반면, 황산염환원의 정도는 50℃ 조건에서 더 크게 나타났다. 철과 황산염이 모두 존재하는 50℃ 조건에서 반응속도가 가장 빠른 것이 확인되었다. 반응의 정도와 속도가 다름에도 불구하고, WRK 벤토나이트의 토착미생물은 두 온도 조건 모두에서 철 및 황산염환원을 할 수 있는 것이 관찰되었다. 한편, WRK 벤토나이트에서 고압멸균환경에서도 생존하고 이후 50℃ 조건에서 지속적으로 대사를 할 수 있는 열 저항성 미생물의 존재 또한 확인되었다. 본 연구 결과를 통해 향후 공학적 방벽물질로 벤토나이트를 선정할 때 온도가 미생물 활동에 미치는 영향을 고려해야 할 필요성을 확인할 수 있었다. The unique geomechanical properties of bentonite, such as swelling capacity that prevents radionuclides leakage, allow this clay mineral to be a promising buffer material for constructing spent nuclear fuel (SNF) repository in deep underground environments. However, indigenous microbes in bentonite can be introduced to the SNF system when the repository is placed in deep underground. The sealing of the repository replaces the ambient environment with anoxic condition; in turn the activities of indigenous microbes such as fermentation and anaerobic respiration may be stimulated and thereby affect the bentonite and by extension, SNF repository stability. In addition, temperature around metal canister, which is the core part of SNF, is increased rapidly by the emitted heat resulting from decay of radionuclides. The temperature change may also induce a variety of microbial activities. Therefore, we deployed microcosms to investigate the temperature effect on the activities of indigenous microbes originated from WRK bentonite at 18℃ and 50℃. Lactate as an electron donor and sulfate and/or ferrihydrite as an electron acceptor were supplied. The results showed that acetate was produced at both 18℃ and 50℃ from lactate whereas only propionate was produced from lactate at 18℃ via early-stage microbial activities. The extent of iron reduction was greater at 18℃ than 50℃, while there was more sulfate reduction at 50℃ than 18℃. The rate of reactions was the fastest under both Fe(III)- and sulfate-amended condition at 50℃. Despite different rate and extent of the reactions, WRK indigenous microbes were able to reduce sulfate and iron under both temperatures. Meanwhile, WRK bentonite also harbored heat-resistant microbes that survived sterilization by autoclaving and continued to metabolize at 50℃. Our findings illustrate the necessity to consider the influence of temperature on microbial activities when employing bentonite as an engineered buffer material for the SNF repository barrier.

Acetate stimulated microbial iron and sulfate reduction and methanogenesis in saturated soil columns affected by dissolution of cement debris

Min, Ha Eun 고려대학교 대학원 2023 국내석사

Construction and Demolition Wastes (CDWs) have become a significant environmental concern due to urbanization and population growth. CDWs are commonly disposed to landfill sites and can generate high pH leachate with various organic and inorganic constituents such as acetate and sulfate, which may affect biogeochemical properties in surrounding environments. Previous studies have mainly focused on changes in geochemical properties and contaminant concentrations. However, the impact of CDW leachate on microbial reactions and communities in subsurface environments remains poorly understood. Here we investigated the continuous impact of CDW leachate on subsurface environments using columns composed of two soil layers: the top layer with cement debris and the bottom layer without cement debris. The columns were fed with artificial groundwater with or without acetate and/or sulfate. The pH of the bottom layer in all columns rapidly increased to 8 - 10 within 28 d. Fe(III)- and sulfate-reduction did not occur in the columns without acetate. However, in the column with acetate alone, Fe(II) was produced with increase in relative abundance of Fe(III)-reducing bacteria followed by increase in methanogenic archaea, Methanosarcina, indicating potential methanogenesis. In the column with both acetate and sulfate, Fe(III) and sulfate were reduced simultaneously along with increase in both Fe(III)- and sulfate-reducing bacteria, and then Methanosarcina appeared at the later time. The results demonstrate that microbial Fe(III)- and sulfate-reduction as well as acetoclastic methanogenesis can occur even in soils with high alkaline pH resulting from the dissolution of cement debris. These results provide insights into the relationship between CDW leachate and subsurface microorganisms and emphasize the need for further research to thoroughly understand the impact of various types of CDW leachates on subsurface biogeochemistry. 건설폐기물은 도시화와 인구 증가로 인해 주요한 환경문제로 대두되고 있다. 건설폐기물은 일반적으로 매립지를 통해 처리되며, 다양한 유기 및 무기 성분을 포함하며 높은 pH 침출수를 생성하며 주변 환경의 생지구화학적 특성에 영향을 미칠 수 있다. 이전 연구는 주로 지화학적 특성과 오염 물질의 농도 변화에 초점을 맞추었다. 그러나 침출수가 지중 환경의 미생물학적 반응과 군집에 미치는 영향에 대해 여전히 잘 알려져 있지 않았다. 따라서, 본 연구에서는 두 개의 토양층으로 구성된 컬럼을 이용하여 침출수가 지중 환경에 미치는 영향을 조사했다. 토양층은 시멘트 잔해를 포함하는 상단 층과 시멘트 잔해가 없으며 매립지 하부 지층을 모사하는 하부층으로 구성되어 있다. 침출수는 아세테이트와 황산염의 유무에 따라 다른 조성의 인공 지하수를 통해 모사하였다. 모든 컬럼의 토양층에서 pH는 28일 이내 8 - 10으로 빠르게 증가하였다. 아세테이트가 없는 컬럼에서는 철 환원과 황산염 환원이 발생하지 않았다. 아세테이트가 있는 컬럼에서는 Fe(II) 농도 증가와 철 환원 박테리아의 상대 풍부도의 증가가 관찰되었으며, 이후 아세테이트를 통해 메탄생성을 하는 고세균인 Methanosarcina가 증가함으로 메탄생성 과정이 일어났음을 보여 주었다. 아세테이트와 황산염을 모두 포함한 컬럼에서는 철 환원과 황산염 환원 박테리아의 상대 풍부도가 동시에 증가하며, 이후 Methanosarcina가 관찰되었다. 연구 결과, 시멘트 파편의 용해로 인한 높은 pH 환경에서도 미생물학적 철 환원, 황산염 환원 및 메탄생성과정이 발생할 수 있음을 보여준다. 이러한 결과는 건설폐기물 매립지에서 발생한 침출수와 지중환경 내 미생물 활동 관계에 대한 통찰력을 제시하며 향후 다양한 조성의 침출수가 지중환경 내 생지구화학적 인자에 미치는 영향에 대한 추가 연구의 필요성을 강조한다.

Effects of Heterogeneous Distribution of Total Petroleum Hydrocarbons on Soil Biogeochemical Properties in Fuel Contaminated Soils from Closed Military Site

Myeong Jung Kang 고려대학교 대학원 2023 국내석사

Liquid fossil fuels, collectively referred to as total petroleum hydrocarbons (TPH), frequently leak into subsurface environments due to the process of being produced, transported, stored and other anthropogenic activities or unexpected accidents. Petroleum-contaminated soil pollutes local groundwater, renders potable water unsafe, limits groundwater use, causes enormous economic loss and ecological disaster, and can destroy agricultural production. As a result, soil contamination by petroleum hydrocarbons brings up serious issues and has potential negative impacts of worldwide environmental and health problem. TPH is highly toxic to organisms, and the regulatory guideline of TPH in soil residential areas in South Korea is 500 mg kg⁻¹. As a clean-up method of TPH in-situ, physical and chemical remediation (e.g., in-situ chemical oxidation using strong chemical oxidants) can relatively quickly remove TPH from soil, but soil organic matter and land productivity can decrease after remediation, which can seriously affect soil ecology. And aerobic TPH biodegradation by injecting oxygen has been most commonly used, but has many limitations because dissolved oxygen has low solubility in water and is consumed quickly by aerobic bacteria. Thus, although anaerobic biodegradation is considerably slower than aerobic biodegradation, this study investigates the potential of anaerobic TPH degradation by indigenous microbes including fermenting bacteria and iron reducing bacteria. The specific objectives of this study are to 1) characterize spatial distribution of TPH in the contaminated site at the closed military base, 2) assess the correlations between TPH contamination and other physicochemical/microbiological properties in contaminated soil, and 3) evaluate the potential of anaerobic TPH biodegradation by indigenous microorganisms including fermenting bacteria and iron reducing bacteria. Twenty soil cores each with 6 to 10 m lengths were taken from a closed military base where TPH has been released from underground storage tanks since 1980s and various physicochemical and microbial properties were determined at every 0.5-meter interval of the cores. We obtained positive correlation between TPH and Fe(II) (Rs=0.414, p<0.001), TPH and total bacterial population (Rs=0.593, p<0.001), and TPH and diesel degrading bacterial population (Rs=0.407, p<0.01). In addition, to assess the relationship between TPH degradation and microbial iron reduction, soil samples were grouped based on the concentration of TPH and Fe(II) [i.e., high TPH (>500 mg kg-1)and high Fe(II) (>450 mg kg-1), high TPH and low Fe(II), low TPH and high Fe(II), and low pH and low Fe(II)]. Alpha diversity was significantly lower in the presence of high TPH regardless of Fe(II) concentrations, suggesting that high concentrations of TPH exerted a strong selective pressure on indigenous bacterial community. In high TPH and low Fe(II) group, fermenting bacteria including Microgenomatia (class) and Chlamydiae (class) were more abundant suggesting that TPH biodegradation is likely occurred via fermentation. In contrast, in high TPH and high Fe(II) group, iron reducing bacteria including Geobacter (genus) and Zoogloea (genus) were more abundant suggesting that TPH biodegradation is likely coupled with microbial Fe(III) reduction. 현대 사회에서 총석유계탄화수소로 통칭되는 액체류 화석연료는 생산, 운반, 저장 및 다른 인간 활동이나 예기치 못한 사고로 인해 지중 환경으로의 누출이 빈번하게 일어난다. 유류로 오염된 토양은 그 지역의 지하수를 오염시키고 음용수와 지하수 사용을 제한하며, 막대한 경제적 손실과 생태학적 위해를 초래하고 농업 생산성을 붕괴할 수 있다. 결과적으로, 총석유계탄화수소에 의한 토양 오염은 심각한 문제를 야기하고 전 세계적인 환경 및 건강 문제에 부정적인 영향을 미친다. 총석유계탄화수소는 유기체에 굉장히 독성이 강하며, 한국 토양주거지역에서의 총석유계탄화수소 규제지침 농도는 500 mg kg-1이다. 총석유계탄화수소의 in-situ 정화 방법으로서 물리적, 화학적 정화 방법 (예를 들어 강한 화학적 산화제를 이용한 in-situ 화학적 산화법) 은 토양에서 총석유계탄화수소를 비교적 빠르게 제거할 수 있지만 정화 후 토양 유기물 및 토지 생산성이 감소하는 등 토양 생태계에 심각한 영향을 미칠 수 있다. 그렇기 때문에 산소를 유류오염 부지에 주입하여 호기성 총석유계탄화수소 생분해를 촉진시키는 방법은 가장 일반적으로 사용되어 왔지만, 용존 산소는 물에 대한 용해도가 낮고 호기성 박테리아에 의해 빠르게 소비되기 때문에 한계점을 가지고 있다. 따라서 본 연구에서는 호기성 생분해보다 발효 미생물, 철 환원 미생물 등 토착 미생물에 의한 혐기 조건에서 총석유계탄화수소의 분해 가능성에 대해 연구하였다. 본 연구의 구체적인 목적은 1) 폐쇄된 군사부지의 오염현장에서의 총석유계탄화수소의 분포 특성 분석, 2) 총석유계탄화수소 오염과 오염된 토양에서의 기타 물리화학적/미생물학적 특성 간의 상관관계 평가, 3) 발효 미생물과 철 환원 미생물 포함하여 토착 미생물에 의한 혐기성 총석유계탄화수소 생분해 가능성을 평가하는 것에 있다. 1980년대 이후 지하 저장탱크에서 유류가 누출된 폐쇄된 군사기지에서 길이 6 ~ 10m의 토양 시추코어 20개에서 0.5 m 간격마다 토양 샘플을 샘플링하여 다양한 물리화학적, 미생물학적 특성을 파악하였다. 총석유계탄화수소와 Fe(II) (Rs=0.414, p<0.001), 총석유계탄화수소와 총 미생물 수 (Rs=0.593, p<0.001), 총석유계탄화수소와 유류 분해 미생물 수 (Rs=0.407, p<0.01) 사이에 양의 상관관계를 얻었다. 또한 총석유계탄화수소 분해와 철 환원 미생물의 관계를 평가하기 위해 토양 시료를 총석유계탄화수소와 Fe(II) 농도를 각각 500 mg kg-1, 450 mg kg-1로 구분 짓고 네 개의 그룹으로 나누었다. 그룹간 미생물 다양성은 Fe(II) 농도에 관계없이 높은 총석유계탄화수소 농도의 그룹에서 낮게 나타났으며, 이는 고농도의 총석유계탄화수소가 토착 미생물 군집에 강한 스트레스 혹은 선택적 압력이 작용했을 것으로 판단된다. 또한 높은 총석유계탄화수소 농도와 높은 Fe(II) 농도 그룹에서 Geobacter (genus) 와 Zoogloea (genus)를 포함한 철 환원 미생물이 더 풍부하게 나타났으며 이는 총석유계탄화수소 생분해가 철 환원 미생물에 의해 일어나고 있음을 시사한다. 이와는 다르게 높은 총석유계탄화수소 농도와 낮은 Fe(II) 농도 그룹에서 Microgenomatia (class) 와 Chlamydiae (class)를 포함한 발효 미생물이 더 풍부하여 총석유계탄화수소 생분해는 발효 미생물에 의해 분해되고 있을 가능성이 있음을 시사한다.