- Title
- Phycoremediation of acid mine drainage
- Creator
- Sudharsanam, Abinandan
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Mining plays a crucial role in the economy of many countries and is one of the primary sources of mineral commodities that are essential in everyday life. The natural process of oxidation (induced by microbial activity or weathering of waste rocks and tailings), lead to the dissolution of pyrite that tends to produce acid mine drainages (AMD). The critical characteristics of AMD are the low pH (2-4) and bioavailability of metals and metalloids including iron, arsenic, cadmium, zinc, cobalt, copper, etc. which at significant levels pose a serious threat to the environment. Conventional treatment involves both active and passive approaches which include the mechanisms such as complexation, sedimentation, and adsorption to immobilize metals and neutralize pH of AMD. However, several limitations, such as excessive chemicals, capital investment, treatment efficiency, and disposal to the environment necessitate the practice of alternative treatment techniques. Microalgae are ubiquitous organisms and some of which can thrive in extreme environments including acid mine drainages are commonly known as acidophiles.,. Besides acquiring innate tolerance to survive in AMDs, the potential of acidophilic microalgae in remediation is very limited. Therefore, this study was aimed to develop green approaches using acid-tolerant microalgae (phycoremediaiton) for cost-effective AMD treatment. Non-acidophilic microalgae species Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3 were found to grow in acid pH with a minimal cell density of 5 × 10⁵ cells mL⁻¹ compared to other reference strains of microalgae. Growth analysis indicated that both the microalgal strains possessed a passive uptake of CO₂ at pH 3.0. Flow cytometry analysis for reactive oxygen species, membrane permeability, and neutral lipids revealed the capabilities of both the strains to adapt to the stress imposed by acidic pH. Lipid production doubled in both the strains when grown at pH 3.0. In-situ transesterification of biomass resulted in 13-15% FAME yield in the selected microalgae, indicating their great potential in biofuel production. Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, isolated from neutral pH environments were investigated for their ability to simultaneously remove heavy metals such as copper (Cu), cadmium (Cd), iron (Fe), manganese (Mn), and zinc (Zn) and produce biodiesel when grown at pH 3.5. Excepting Cu, the selected metals at concentrations of 10−20 mg L⁻¹ supported good growth of both the strains. MAS1 was tolerant even to 20 mg L⁻¹ of Cd while strain MAS3 could withstand only up to 5 mg L⁻¹. Cellular analysis for metal removal revealed the predominance of intracellular mechanism in both the strains resulting in 40−80 and 40−60% removal of Fe and Mn, respectively. FTIR analysis revealed that the symmetric vibrational stretch observed in Amide-I region (1650-1670 cm⁻¹) increased with increasing concentrations of metals in MAS1, but not in MAS3. However, the symmetric and asymmetric vibrational stretches characteristic of carboxylic esters (1735-1750 cm⁻¹) and methylene groups (2925 and 2960 cm⁻¹) of lipids were significant in strain MAS3 than in strain MAS1. FTIR analysis of metal-laden biomass indicated the production of substantial amounts of biodiesel rich in fatty acid esters. Sustainable resource recovery is the key to manage the overburden of various waste entities of mining practices. The present study demonstrates for the first time a novel approach for iron recovery and biodiesel yield from two acid-adapted microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, grown in synthetic acid mine drainage (SAMD). Virtually, there was no difference in growth of the strain MAS3 both in Bold’s basal medium (control) and SAMD. Using IC50 level (200 mg L⁻¹) and a lower concentration (50 mg L⁻¹) of iron in SAMD, the cell granularity, exopolysaccharide (EPS) secretion, iron recovery, and biodiesel were assessed in both the strains. Both cell granularity and accumulation of EPS were significantly altered under metal stress in SAMD, resulting in an increase in total accumulation of iron. Growth of the microalgal strains in SAMD yielded 12‒20% biodiesel, with no traces of heavy metals, from the biomass. The entire amount of iron, accumulated intracellularly, was recovered in the residual biomass. Our results on the ability of the acid-adapted microalgal strains in iron recovery and yield of biodiesel when grown in SAMD indicate that they could be the potential candidates for use in bioremediation of extreme habitats like AMD. Phenotypic plasticity or genetic adaptation in an organism provides phenotypic changes when exposed to the extreme environmental conditions. The resultant physiological and metabolic changes greatly enhance the organism’s potential for its survival in such harsh environments. In the present novel approach, we tested the hypothesis whether acid-adapted microalgae, initially isolated from non-acidophilic environments, can survive and grow in acidmine-drainage (AMD) samples. Two acid-adapted microalgal strains, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, were tested individually or in combination (co-culture) for phenotypic changes during their growth in samples collected from AMD. The acid-adapted microalgae in AMD exhibited a two-fold increase in growth when compared with those grown at pH 3.5 in BBM up to 48h and then declined. Furthermore, oxidative stress triggered several alterations such as increased cell size, granularity, and enhanced lipid accumulation in AMDgrown microalgae. Especially, the apparent limitation of phosphate in AMD inhibited the uptake of copper and iron in the cultures. Interestingly, growth of the acid-adapted microalgae in AMD downregulated amino acid metabolic pathways as a survival mechanism. This study demonstrates for the first time that acid-adapted microalgae can survive under extreme environmental conditions as exist in AMD by effecting significant phenotypic changes. Acid mine drainage (AMD) resulting from mining activities is a serious threat to the environment affecting terrestrial and aquatic life. In this study, acid-adapted microalgae, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, were assessed for their ability in iron (Fe) removal from acid mine drainage samples using varying cell densities in non-immobilized and immobilized systems. Use of free-cells and immobilized cells exhibited 46-48% and 65-79% Fe removal, respectively, after 48 h of incubation. Flow cytometry analysis revealed significant changes in morphology (FSC) and granularity (SSC) in non-immobilized cells than in cells immobilized in alginate beads exposed to AMD samples. Second derivative spectra from Fourier transform infrared (FTIR) spectroscopy revealed vibration stretching for polysaccharides (1094 cm⁻¹) of free-cells, and protein (1500-1700 cm⁻¹), hydroxyl (3000-3500cm⁻¹) of immobilized cells as a protective mechanism against Fe present in AMD samples. Group clustering of variables of free-cells and immobilized cells of microalgal strains was very evident as revealed by principal component analysis. Artificial neural network modelling validated the experimental data obtained in column studies with R2 >0.95. Fixed bed column was conducted using two different bed depths of 1 cm and 2 cm with immobilized microalgal strains of MAS1 and MAS3 achieved a greater breakthrough time for 2 cm bed height, indicating the requirement for an extended contact time of the adsorbate to the adsorbent. The present study demonstrates the application of microalgal cells entrapped in alginate beads in batch and column in a greener and economical approach to treat AMD for sustainable mining. All these experiments comprehend the effectiveness of acid-tolerant microalgae MAS1, and MAS3 as promising candidates for AMD remediation. Further, the studies also revealed possible recovery of metal (Fe) from the residual biomass after biodiesel extraction.
- Subject
- non-acidophilic microalgae; acid adaptation; acid mine drainage; resource recovery; bioenergy; sustainable mining; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1418259
- Identifier
- uon:37319
- Rights
- Copyright 2020 Abinandan Sudharsanam
- Language
- eng
- Full Text
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