Graduation Year


Document Type




Degree Name

Master of Science (M.S.)

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Valerie Harwood, Ph.D.

Co-Major Professor

Jeffrey Cunningham, Ph.D.

Committee Member

Kathleen Scott, Ph.D.


Aspergillus niger, Penicillium chrysogenum, Penicillium simplicissimum, Organic Acids, Cathode


Demand for lithium (Li) and cobalt (Co) is on the rise, due in part to their increased use in rechargeable Li-ion batteries (RLIB). Current recycling processes that utilize chemical leaching efficiently recover in Li and Co from the cathode material in spent batteries; however, these processes are costly and emit hazardous waste into the environment. Therefore, a more sustainable process for recycling Li and Co is needed, and bioleaching may provide a solution. Fungal bioleaching has been shown in previous studies to effectively mobilize metals (Pb, Al, Mn, Cu, and Zn) from mine tailings, electronic scrap, and spent batteries with organic acids. However, little is known regarding fungal tolerance to Li and Co, and if the concentrations of organic acids excreted by fungi can effectively leach Li and Co from the cathode material.

In order to address these questions, experiments were first conducted to test the Li and Co leaching efficiency with organic acids at concentrations similar to what has been previously reported in fungal cultures. The remaining experiments were performed with three fungal species: Aspergillus niger, Penicillium chrysogenum, and Penicillium simplicissimum. First, fungal biomass production, pH and organic acid excretion were examined when the fungi were grown in Czapek dox broth (CDB) or Sabouraud dextrose broth (SDB). Second, fungal biomass production and pH were examined when the fungi were grown in the presence of Li or Co. This determines tolerance of the fungi to the metals, and if fungal processes were inhibited by the metals. Third, bioleaching was performed with cathode material from RLIB in batch cultures to test the ability of organic acids excreted by A. niger to mobilize Li and Co. Three bioleaching strategies, one-step, two-step, and spent-medium leaching techniques were used to mobilize Li and Co from the cathode in RLIB.

Low concentrations of organic acids similar to what is excreted by fungi have not been tested to leach Li and Co from the cathode in RLIB. Results from chemical leaching with low concentrations of organic acids in this study indicate that organic acid leaching efficiency can be increased by utilizing higher concentrations (above 50 mM) of citric or oxalic acid to mobilize Li or Co from the cathode in RLIB. Furthermore, 100 mM of citric acid or 100 mM of oxalic acid mobilized more Co or Li than mixtures of organic acids. Notably the addition of hydrogen peroxide to mixed concentrations of organic acids significantly improved mobilization of Li and Co under abiotic conditions.

Different growth media may alter biomass production and potentially organic acid excretion by the three fungal species. Analysis of biomass production by A. niger and P. simplicissimum showed that differences in media composition between CDB and SDB did not affect collected biomass for each species. However, CDB cultures with P. chrysogenum had significantly less biomass than SDB cultures after 10 days of growth. Differences in growth by P. chrysogenum between CDB and SDB may be attributed to preferred nutrients and/or low pH present in SDB cultures. Biomass production by the three fungi increased up to day 10 in CDB or SDB. This result indicated that nutrients in CDB or SDB were not limiting toward fungal growth. Cultures with A. niger had the highest concentrations of organic acids (50 mM of oxalic acid), followed by cultures with P. simplicissimum (30 mM oxalic acid), and P. chrysogenum (less than 5 mM oxalic acid). Organic acids excreted by all three fungal species were detected in cultures in CDB, while only A. niger and P. chrysogenum excreted organic acids in SDB cultures.

Metals such a Li or Co present in the cathode of RLIB may be toxic to fungal processes when exposed to high metal concentrations. Metal tolerance experiments indicate that biomass production by the three fungi was significantly inhibited by 100 mg/L Co compared to controls, which contained no metal. Li at a concentration of 1000 mg/L inhibited biomass production by A. niger and P. simplicissimum. However, biomass production by P. chrysogenum was not significantly inhibited by 1000 mg/L Li. I found that P. simplicissimum was the most susceptible to toxic effects of Li and Co among the three fungi. In A. niger cultures amended with 100 mg/L Li or Co, pH at day 5 was similar to control cultures of A. niger without metals (pH 3.0 – 3.4), whereas pH was significantly higher in cultures with 1000 mg/L of Li or Co (pH 7.1 – 7.3).

Cultures of A. niger were exposed to the cathode material from RLIB to test the leaching efficiency of excreted organic acids after mobilizing Li and Co. In bioleaching experiments with A. niger, organic acids excreted in the presence of cathode material from RLIB were quantified at concentrations under 50 mM. At the end of bioleaching experiments with A. niger, 40 mM tartaric acid was detected and was the highest produced organic acid in bioleaching cultures. However, with conditions set in this study, organic acids excreted by A. niger mobilized only ̴7% of Co and 20% of Li when using spent medium with cathode material from RLIB. According to findings in chemical leaching experiments, concentrations of organic acids higher than 50 mM will be required in fungal cultures to increase mobilization of Li or Co from the cathode material in RLIB. Modifying growth media to include higher concentrations of sucrose will potentially increase organic acid excretion as demonstrated in previous publications. Future studies should focus on how to maximize organic acid excretion by fungi when exposed to metals found in the cathode of RLIB.