Strategies to stimulate biosulfidogenesis in the deep layer of a meromictic acidic pit lake for environmental remediation
Open Access
- Author:
- Liu, Yutong
- Graduate Program:
- Environmental Engineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 24, 2025
- Committee Members:
- Jay Regan, Professor in Charge/Director of Graduate Studies
William Burgos, Co-Chair & Dissertation Advisor
Jay Regan, Major Field Member
Jennifer Macalady, Co-Chair, Outside Field Member & Dissertation Advisor
Rachel Brennan, Major Field Member
Estelle Couradeau, Outside Unit Member - Keywords:
- Acidic pit lake
Bioremediation
Heavy metal(loid)s
Sulfur cycle
Sulfate reducing bacteria
Microbiology - Abstract:
- Acidic pit lakes are formed by the flooding of abandoned open pit mines, where the polymetallic sulfide ores are exposed to water, and oxidants, such as oxygen (O₂) and ferric iron (Fe³⁺), catalyze their oxidative dissolution. They posed significant environmental challenges over the past few decades, particularly after periods of intense mining activity and the closure of mining sites without proper protection and restoration measures, resulting in the collection of significantly metal(loid)-rich and acidic water in open ponds. The water in acidic pit lakes is widely regarded as a pressing environmental issue requiring remediation. This Ph.D. research focused on studying processes occurring in the deep layer of the well-studied meromictic acidic pit lake, Cueva de la Mora (CM), located in Huelva Province in southwestern Spain. Chapters 2 and 3 focused on strategies for the removal of harmful metal(loid)s and the neutralization of lake water. Traditional remediation methods primarily employ alkaline addition, such as soda ash or lime, to raise pH and immobilize such metal(loid)s by forming metal hydroxide and/or metal carbonate precipitates. However, this approach can be expensive and generate secondary pollutants, such as bulky sludge (Johnson et al., 2019). Herein, we stimulated biogenic dissimilatory sulfate reduction (biosulfidogenesis) as an alternative approach to immobilize harmful metal(loid)s by precipitating them as low-solubility metal(loid)–sulfide compounds. A challenge with this approach, however, is the lack of electron donors in the deep layer of CM to facilitate dissimilatory sulfate reduction. Furthermore, although previous research has revealed the presence of sulfate-reducing bacteria (SRB) and genes regulating dissimilatory sulfate reduction in the deep layer of CM, the relative abundance of SRB is below 1%, and the gene abundance is lower than that of other major metabolic pathways. This leads to uncertainty regarding the growth of SRB to functionally significant levels. Chapter 2 aimed to promote biosulfidogenesis by supplying substrates that are lacking in the deep layer of CM in a lab-based study. Glycerol was selected as the organic substrate and electron donor, demonstrating its effectiveness in promoting biosulfidogenesis in this layer. The combined addition of glycerol and elemental sulfur (S(0)) further increased sulfide production rates and enhanced the removal of metal(loid)s. This suggested that acid-tolerant sulfate-reducing microorganisms in the deep layer of CM can also utilize S(0) as an electron acceptor to produce sulfide, provided that suitable electron donors are available. Notably, sulfur disproportionation did not occur in the presence of S(0) on its own. From a microbiological perspective, the addition of glycerol enriched the acid-tolerant SRB genus Desulfosporosinus (76%-96% relative abundance); even more efficient sulfide production were observed in microcosms containing both glycerol and S(0), along with a higher enrichment of Desulfosporosinus (93-99%). This chapter was accepted for publication in Frontiers in Microbiology in September 2024. Chapter 3 evaluated high-density biomass as the substrate and electron donors to stimulate biosulfidogenesis in the deep layer of CM in a lab-based study. Solid-phase amendments were selected because they can be pressed into pelletized form that is dense enough to settle into the deep layer of a stratified lake. This ‘direct delivery’ of electron donor overcomes the current ‘indirect method’ to stimulate algae growth in the upper layer of the lake and wait for it to die and settle into the deep layer. Solid-phase amendments tested were two acid-tolerant microalgae and one aquatic plant. Coccomyxa, the dominant microalgal genus in the surface layer and chemocline of CM, was introduced in dry powder form as the organic substrate. Additionally, other types of solid-phase biomass, including the acid-tolerant microalgae Euglena, and duckweed (Lemnoideae minor) were evaluated. All types of solid-phase biomass tested promoted the growth of acid-tolerant SRB and enhanced sulfide production. Biocomponents of these complex amendments were assessed to better understand the stimulatory effects of amino acids, sugars, and long-chain fatty acids. Amino acids emerged as the preferred biocomponent for sulfate reduction in the deep layer of CM, based on the shorter adaptation period, while sugar monomers demonstrated a sulfide production rate comparable to that of amino acids once dissimilatory sulfate reduction commenced. Chapter 4 addressed another potential limiting factor leading to low sulfide accumulation and metal(loid) removal in the deep layer of CM: the suspected continuous input of trace amounts of oxygen due to groundwater recharge. According to the chemical profiles of this lake, sulfide was produced and accumulated under strictly anoxic conditions but underwent biogenic re-oxidation upon the introduction of trace oxygen. Simultaneously, metal(loid)s, particularly arsenic, which had been immobilized under anoxic conditions, re-dissolved upon oxygen exposure. Biologically, the acid-tolerant SRB genus Desulfosporosinus was significantly enriched under anoxic conditions but its growth was inhibited under oxic conditions. In contrast, the acid-tolerant sulfur-oxidizing bacterial genus Acidithiobacillus proliferated under oxic conditions, increasing in abundance with an increasing number of redox oscillation phases. Genetic analysis via quantitative polymerase chain reaction (qPCR) revealed the high abundance of dissimilatory sulfate reduction genes under anoxic conditions but their inhibition upon the introduction of trace amounts of oxygen. Conversely, sulfur-oxidizing genes remained undetected until oxygen was introduced, and their abundance gradually increased with prolonged oxygen exposure. In conclusion, this research demonstrated that biogenic sulfur oxidation, stimulated by the continuous input of trace amounts of oxygen, is a critical factor limiting sulfide accumulation and, consequently, suppressing metal(loid) removal. To the best of our knowledge, this is the first laboratory study investigating the complete S cycle in acidic pit lakes.