Analysis of Sulfide Inhibition in Anaerobic Biofilm Reactors Fed a Sulfate-Rich Wastewater

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Introduction
Many industries produce wastewaters high in organic and sulfate content. Anaerobic biological treatment of these wastewaters can result in the formation of methane by methanogenic archaea (MA) and sulfide as sulfate reducing bacteria (SRB) reduce the sulfate to sulfide. Unfortunately, sulfide can inhibit the activity of both SRB and MA. Previous research has shown that biofilm processes can withstand higher levels of sulfide, while suspended growth systems showed deteriorating performance at much lower levels. The advantage for biofilm reactors is not well understood. This research hopes to help increase our understanding of how biofilms develop this resistance to sulfide toxicity when treating wastewaters containing sulfate.

Materials and Methods
Three reactors were constructed (ID=3”, length=36”) with pumice as a support material and seeded with anaerobic enrichment cultures (2.6L each). They were fed a mineral media containing sulfate and lactate. Reactors were kept at 21ºC and the hydraulic retention time (HRT) was two days. Samples were regularly taken for analysis of chemical oxygen demand (COD), volatile fatty acids (VFAs), sulfide, sulfate, methane, and pH. DNA was extracted from pumice samples removed from the reactors on day 180 and a clone library was constructed for Reactors 1 (R1) and 2 (R2) in order to determine what kinds of SRB and MA were present in the reactor.

Results and Discussion
The influent COD of the reactors was increased from 600 mg/L to a maximum of 7700 mg/L. While R2 and Reactor 3 (R3) had effluent COD levels below 100 mg/L at the maximum, R1 showed increased effluent COD of around 900 mg/L at a load of 6500 mg/L COD. Meanwhile, gas composition started around 80% methane then decreased and remained relatively constant, with a 700 day average for all three reactors of 53% ± 13% methane.

While feeding a high influent COD of 7700 mg/L, reactors were spiked with lactate to observe the effect of a lactate shock. R1 showed the least perturbation, with effluent COD increasing 500 mg/L, but decreasing back to around 1000 mg/L within 35 days. R2 and R3 COD levels went from zero to 3000 mg/L and did not decrease until influent COD was decreased by half 30 days after the spike .

Sulfate levels were also increased over time from 17 mg-S/L to a maximum of 1000 mg-S/L. As feed sulfate increased, sulfate removal averaged about 95% and effluent sulfide increased to about 500 mg-S/L. When feed sulfate reached 1000 mg-S/L, but before the lactate spike, sulfate removal started to decrease in all reactors. Sulfide levels continued to be high, however. The lactate spike resulted in a decrease in effluent sulfide in R2 and R3 from around 500 mg-S/L down to zero, although recovery to previous sulfide levels did eventually occur after about 60 days. R1 sulfide levels were affected less by the lactate spike, with levels decreasing from around 600 mg-S/L to 200 mg-S/L with recovery after about 25 days. Reactor operation data appears to show that increasing sulfide levels and lactate spiking did not affect MA activity but did negatively affect SRB activity.

Clone library results showed that the main MA clones were similar to the acetate utilizer Methanosaeta concilii. A majority of SRB clones were similar to Desulfovirbio and Desulfomocrobium groups, and clones similar to Desulfobulbus and Desulfococcus were found.

Future and Ongoing Work
Additional DNA will be extracted from the reactors. A clone library will be constructed and compared to the clone library made previously. Also, quantitative PCR (qPCR) will be used on both DNA sample sets in order to determine the relative quantities of different SRB and MA. By comparing the clone library and qPCR results during different operational conditions, the effect of different organism groups on aggregate reactor behavior will be seen.

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