The effect of carbon inputs on microbial community structure and function: the role of fermentation processes in groundwater.

Abstract

Carbon inputs to groundwater aquifers include intentional applications, as in bioremediation practices, and unintentional spills. The addition of carbon to an aquifer environment promotes the growth of a diverse and complex microbial community capable of generating several fermentation products, including some regulated compounds and methane, an explosive gas. This dissertation focuses on the fermentative community that develops in response to carbon application in an aquifer environment. Research was conducted to specifically examine 1) how fermentation processes affect partitioning of trichloroethene (TCE), a common groundwater contaminant, 2) the extent that continuous or pulsed carbon inputs affect microbial community structure and function, and 3) how an ethanol-based fuel (E85) stimulates fermentation processes, including methane generation, and the effect of ethanol toxicity on plume longevity. Remediation of groundwater plume source areas is challenging because lingering contaminants are often present as non-aqueous phase liquid (NAPL) and sorbed mass, and therefore difficult to remove via biodegradation or other commonly used remedial methods. Experimental results indicated that enhanced dissolution of TCE NAPL was possible through the addition and/or subsequent fermentation of a dilute molasses solution. Two mechanisms were responsible for the enhanced dissolution of NAPL; the addition of fresh molasses increased TCE solubility (>200%), thereby increasing the concentration gradient and subsequent mass transfer of NAPL to the dissolved phase, and mixing NAPL with fermented molasses solution significantly increased the surface area of the NAPL through formation of an emulsion, thereby increasing the mass flux of NAPL to the dissolved phase. In addition, the fermented liquid may have also decreased the soil partitioning coefficient (Kd) of TCE, indicating that enhanced transfer of sorbed mass to the aqueous phase could also occur in the presence of fermented molasses. These results can be used to optimize remedial systems to increase NAPL and sorbed-mass dissolution and are therefore important, particularly when bioremediation is used to polish residual source zones. The addition of organic carbon to a groundwater aquifer for biostimulation purposes promotes the growth of a diverse fermentative community as well as organisms targeted for contaminant degradation. Engineered carbon application systems commonly include either a continuous low dose of carbon, or periodic high doses of carbon. Experimental results indicated that a monthly pulse of a high dose (10% by volume) of molasses generated several fermentation products at high levels following each application, while a continuous feed of low molasses solution (0.4%) reached steady-state in 130 days, after which no further detection of fermentation products occurred. Methane generation in both systems was similar, indicating that methane production was not affected by the carbon addition strategy. Significant shifts in both Eubacteria and Archaea community structures were observed after carbon introduction, with the greatest changes correlating to the higher concentrations of carbon provided by the pulsed system. The total quantity of bacteria and methanogens was higher along the pulsed-fed column compared to the continuously-fed system. The continuously-fed column exhibited greater biofouling behavior. Taken together, biofouling did not appear to be a result of biomass quantity, rather a function of community structure. In summary, the method of carbon introduction (pulsed high-dose versus continuous low-dose) can result in significantly different community structures, functions, and densities of indigenous organisms. These data suggest that systems can be engineered to control fermentation product generation and biofouling behavior by manipulating the style of carbon application. Methane, however, will need to be controlled in either system. A spill of ethanol-based fuel will not only contaminate an aquifer, but will also serve as a food source to stimulate fermentative organisms that can generate potentially regulated compounds and create an environment conducive for production of explosive methane gas. Experimental results indicated that a continuous supply of a dilute ethanol-based fuel (E85) resulted in a profound shift in the community structure of Eubacteria and Archaea accompanied by the production of volatile fatty acids and butanol, a compound with a groundwater regulatory standard in Minnesota. Data also indicated that dissolved methane was produced at concentrations that could accumulate to an explosive level (>2 mg/L) in headspace. Quantitative polymerase chain reaction (qPCR) data showed a statistically significant increase in methanogenic populations, when compared to a control column. These results strongly correlated to areas of the column containing acetate, a breakdown product of ethanol. Toxicity data indicated that microbial growth was completely inhibited at approximately 6% (vol/vol) ethanol. These results suggest that even though ethanol is readily degradable, the core of an E85 spill may serve as a long-term source of contamination, and subsequent methane production, as it cannot be degraded until significant dilution has occurred. The research presented in this dissertation shows that the addition and subsequent fermentation of molasses can enhance the mass transfer of TCE, and that the style of carbon application affects the microbial community structure, density of biomass, and subsequent production of fermentation processes. Similarly, an input of E85 will result in the generation of fermentation products, some of which are regulated, and produce methane at levels that can potentially accumulate to explosive levels. This research furthers our understanding of the importance of fermentation processes resulting from carbon inputs to a groundwater environment. These results can be used to optimize bioremediation systems that incorporate carbon addition in order to manage fermentation product formation and biofouling impacts, and to mitigate potential human health hazards stemming from ethanol-based fuel spills through more accurate fate and transport modeling efforts.