Masters Theses

Xiaoyan Zhang

Date of Award

8-1998

Degree Type

Thesis

Degree Name

Master of Science

Major

Environmental Engineering

Major Professor

Kevin G. Robinson

Committee Members

Chris Cox, Gregory Reed

Abstract

Anaerobic microbial reduction of soluble hexavalent uranium (U (VI)) has been reported for iron and sulfur reducing bacteria. A facultative anaerobic iron reducer, Shewanella alga is considered one of the most promising strains for uranium biotreatment because it is easy to grow and can effectively reduce U (VI). The purpose of this research is to further evaluate the potential of this organism in uranium waste biotreatment. The research goals include investigating possible growth of S. alga using U (VI) as the sole electron acceptor and exploring the impact of iron on uranium reduction under both non-growth and growth conditions. Metal reduction and microbial growth kinetics were also studied and the kinetic coefficients determined to predict the performance of S. alga for metal reduction.

All batch tests were conducted in duplicate using 100 mL serum bottles. The seed S. alga culture was grown in an anaerobic medium containing ferric citrate as the electron acceptor. U (VI) and/or Fe (III) were introduced into test solutions as terminal electron acceptors. Each test solution contained 2.5 g/L of sodium bicarbonate (pH buffer) and 2mL/L of 60% w/w lactate syrup (equivalent to 20 mM lactate) as the electron donor. For growth coupled studies, a chemically defined medium which included nutrients essential for microbial growth was used. The initial Fe (III) and/or U (VI) concentrations used were ten times higher than those used in non-growth studies in order to support measurable cell mass. All experiments were conducted at constant pH (8.4) and temperature (35°C).

The Monod model was used to quantify the kinetic data for Fe (III) and U (VI) reduction under non-growth conditions. Results show that the maximum specific reduction rate for Fe (III) was ~5.13 /hr, more than ten times higher than that for U (VI) ( ~0.39 /hr). The half-saturation constant for Fe (III) was ~36.8 mg/L, two times lower than that for U (VI) ( ~ 74.04 mg/L). This suggests that S. alga has a higher reduction capability and a higher enzymatic affinity for Fe (III) than U (VI) when lactate serves as the electron donor.

When both metals were present in test solutions under non-growth conditions, S. alga preferentially reduced Fe (III). This was most likely because Fe (III) reduction does not require as low a redox potential as U (VI) and was therefore more thermodynamically favorable. Subsequent U (VI) reduction after significant Fe (ill) reduction appeared to be impeded by poising the electron potential caused by the [Fe (II)/Fe (III)] redox couple. At low initial Fe (III) concentrations, U (VI) reduction occurred slowly after Fe (III) reduction, suggesting a weak Eh buffering effect. At high initial Fe (III) concentrations, no U (VI) reduction was measured during the experimental duration. It's believed that, high initial Fe (III) levels produced high [Fe (Il)/Fe (III)] ratios which result in strong Eh buffering. Such buffering may poison the system by keeping the Eh value higher than that required for U (VI) reduction. The presence of U (VI) had no impact on Fe (III) reduction kinetics. Tests also showed that Fe (II) initially added had a negative impact on U (VI) reduction.

Growth of S. alga using U (VI) as the sole terminal electron acceptor was demonstrated under batch conditions. Unlike D. desulfuricans, S. alga grew in a minimum salt medium with lactate as the electron donor/carbon source and U (VI) as the electron acceptor. The rate of microbial growth was four times higher than that previously reported for another iron reducer, Geobacter metalireducens. The maximum specific growth rate was estimated to be 0.0215 /hr for U (VI) reduction, and 0.139 /hr for Fe (III) reduction.

Growth-coupled reduction was more complicated because active microbial activities, such as oxidative phosphorylation and cellular synthesis/division, were involved (Bailey and Ollis, 1986). The individual reduction rate of either metal was slower when both metals were present in the medium compared to the rate when one of them was absent. The impact of one metal on the initial reduction rate of the other was both concentration dependent. A combination of initial concentrations of 10 mM Fe (III) and 5 mM U (VI) impeded 90% of the reduction of both metals. Unlike non-growth results, reduction of U (VI) occurred during iron reduction when coupled with microbial growth. In addition, the presence of U (VI) inhibited iron reduction. The reasons are believed associated with growth mechanisms.

Similar to G. metalireducens and Shewanella putrefaciens, S. alga grew when U (VI) served as the sole electron acceptor when lactate was the electron donor. But unlike D. desulfuricans, which utilized two different enzyme systems for sulfate and uranium reduction, the reduction of U (VI) and Fe (III) by S. alga seemed to share the same active enzyme. Under non-growth conditions, the presence of Fe (III) delayed U (VI) reduction. The Fe (Il)/Fe (III) redox couple subsequently inhibited U (VI) reduction by keeping the electron potential of the medium higher than that required for the reaction to occur. While under growth conditions, the reduction of U (VI) and Fe (III) occurred simultaneously. However, reduction was slower and incomplete for each metal compared to that when one of them was absent.

The significance of growth using U (VI) as the sole electron acceptor by S. alga is important. Combined with other advantages of this bacterium, such as the high tolerance to oxygen, the high uranium reduction rate, and no sulfide co-precipitation problems, a continuous flow treatment process using this organism appears promising for selective reduction of U (VI) at a high rate.

Regarding results in the presence of both electron acceptors, predictions could be made on iron containing uranium wastewater treatment. A delay in uranium reduction is expected if S. alga cell suspensions are used. The duration of the delay is Fe (ill) concentration dependent. Above certain iron concentrations, the iron redox couple could possibly poise the system such that U (VI) reduction could never occur. However, U (VI) reduction will occur slowly, although incompletely, when cells are placed in a growth medium. It's suggested that for a uranium waste containing high levels of sulfate and low levels of iron (less than 0.2 mM), S. alga, rather than D. desulfuricans should be utilized.

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