B. Kim1*, J. Novitch1, A. Ontiveros-Valencia2,, M. Vega1, R. Nerenberg1
1 University of Notre Dame, Notre Dame, IN, USA
2 Instituto Potosino de Investigacion Cientifica y Tecnologica, Camino a Presa de San Jose, Mexico
* Bum Kyu Kim: [bkim13@nd.edu]
Summary:We propose a novel type of biofilm reactor where biofilms grow on microfiltration membranes that supply aqueous substrates or performance enhancing chemicals. We carried out two tests as proof of concepts.
In the first, the membranes were used to supply chlorate to the base of a denitrifying biofilm to enrich for perchlorate-reducing bacteria. In the second, cycloheximide, a eukaryotic inhibitor, was supplied to the base of a nitrifying biofilm to suppress protozoan predation. In the first study, chlorate reduction potential increased during the first few days of supplying chlorate, suggesting an enrichment for chlorate and perchlorate-reducing bacteria.
Also, DNA sequencing analysis showed an enrichment for putative perchlorate-reducing bacteria. In the second, cycloheximide supplied to the base of a nitrifying biofilm showed less protozoa and a greater biofilm thickness than a control without cycloheximide. Together, these results show the potential for using water-permeable membranes to supply performance-enhancing chemicals to the base of the biofilm, rather than the bulk. Supply to the base greatly reduces the amount of chemicals required, as compared to supply from the bulk. It also minimizes loss of the chemical with the effluent, and in some cases may be more effective. Further research is needed to explore the practical implementation of this technology.
Keywords: Counter-diffusional membrane; microfiltration membrane; Nitrifying biofilms; Denitrifying biofilms;
Protozoa
Introduction
The hollow-fiber membrane biofilm reactor (MBfR) is an emerging technology used to transfer gases substrates, such as O2 and H2, to the base of biofilms growing on the membrane’s outer surface [1-3].
A major advantage of MBfRs is they supply substrates directly to the base of the biofilm, bypassing the bulk liquid. Supplying substrates to the bulk would require higher concentrations in order to penetrate into the biofilm, and also would lead to the loss of chemicals with the effluent. Despite these advantages, the MBfR is limited, as it only can supply gaseous substrates.
In this study, a novel biofilm reactor was used to overcome the limitation of MBfRs. The proposed reactor uses microfiltration membranes to supply aqueous chemicals.
Two types of experiments were conducted. In one, the membranes were used to supply chlorate to the base of a denitrifying biofilm to enrich for perchlorate-reducing bacteria. Most perchlorate reducing bacteria also reduce chlorate, and most are also denitrifiers. Adding chlorate to the influent of a denitrifying biofilm reactor has been previously shown to select for perchlorate-reducing bacteria[4].
In the second experiment, cycloheximide, a eukaryotic inhibitor, was supplied to the base of a nitrifying biofilm to suppress protozoan predation. Protozoa cause biofilm loss, and also can promote biofilm detachment by weakening the base of the biofilm [5 and 6]. The suppression of protozoa could lead to thicker nitrifying biofilms with increased contaminant removal rates.
Materials and methods
In the first experiment, denitrifying biofilms were grown on the surface of microfiltration membranes.
About 300 mgCOD/L of acetate (i.e., electron donor) was fed through the bulk and about 25 mgN/L of nitrate was supplied through the membrane lumen. Normally, nitrate would be supplied with bulk influent, but for this research it was supplied through the membrane to minimize suspended growth.
140 The reactor had 80 cm of microfiltration membrane (Suez, ZeeWeed, Suez) with an outside diameter of 20 mm. The reactor HRT was 12 hours. After a stable biofilm was formed, 10 mgCl/L of chlorate, together with the nitrate, was added through the membrane lumen. The effluent COD, nitrate, and chlorate were measured daily to track performance. Molecular analysis with Illumina sequencing of the 16S rRNA genes was used to confirm the shift of bacterial community.
For the second experiment, nitrifying biofilms were grown in the same reactor. 30 mgN/L of ammonium ion was supplied through the lumen and PBS medium was supplied in the bulk. Air was sparged in the bulk to maintain the DO. Two reactors were studied. In one, cycloheximide at 500 mg/L (≅1025 mgCOD/L) was supplied through the membrane lumen along with ammonium. Second was run as a control without cycloheximide. The effluent ammonium, nitrate and nitrate were measured daily. Also, OCT images were taken to assess biomass accumulation.
Results and discussion
For the first experiment, prior the addition of chlorate, the steady-state effluent COD and nitrate was 55 mgCOD/L and 0.2 mgN/L, respectively. This showed that the reactor could successfully denitrify. With the addition of chlorate, the steady-state effluent COD was around 10 mgCOD/L, which suggests that chlorate was being consumed. Suspended solids were minimal before and after chlorate addition, below 5 mgVSS/L. Bulk chlorate concentrations were below the detection limit (around 0.1 mg/L). Batch tests with chlorate confirmed chlorate consumption. The 16S rRNA gene sequencing also suggested that the abundance of putative perchlorate-reducing bacteria (e.g., Dechlorosoma) increased with the addition of chlorate (Figure 1).
For the second experiment, the steady-state effluent ammonium was around 10 mgN/L without suppression of protozoa (control). With cycloheximide addition, the steady-state effluent ammonium concentration was around 2 mgN/L. This suggests that suppression of protozoa can increase ammonium removal rates. The OCT analysis showed that the biofilm thickness was substantially greater with cycloheximide addition. Typical OCT images are shown in Figure 2. A more comprehensive analysis of biofilm thickness will be provided in the presentation. For cycloheximide, the bulk concentrations were not directly measured, but the increase in effluent COD with cycloheximide addition was negligible, suggesting very low levels in the bulk.
The proposed microfiltration membrane biofilm reactor was shown to impact biofilm performance without directly adding chemicals to the bulk. By supplying them to the base of the biofilm, bacteria in the biofilm were exposed to higher concentrations, with minimal amounts were released to the bulk liquid.
Mass transfer of chemicals from the microfiltration membrane to the biofilm can occur by diffusion or advection. Most likely the dominant mass transfer mechanism was advection. Selecting the proper advective flux through the membrane is important to preventing loss to the bulk liquid. This flow is likely dependent on the pressure difference across the membrane, the membrane mass transfer resistance, and also the permeability of the biofilm. Uneven biofilm coverage could led to “leakage”
into the bulk. Further research is needed to better understand these factors.
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Figure 1. Results of the microbial community analysis of the denitrifying reactor, at the genus level. Samples S-01,02 and 03 are replicates for the denitrifying biofilm prior to chlorate addition. S-04 is the inoculum. S-05,06, and 07 are replicates for the condition with chlorate addition. The increase of Dechloromonas and Dechlorosoma suggest an enrichment for perchlorate-reducing bacteria.
Figure 2. Typical OCT image of nitrifying biofilms. (A) control, and (B) with cycloheximide
References
[1] Nerenberg, R., and B. E. Rittmann. "Hydrogen-based, hollow-fiber membrane biofilm reactor for reduction of perchlorate and other oxidized contaminants." Water Science and Technology 49.11-12 (2004): 223-230.
[2] R. Nerenberg (2016). The Membrane Biofilm Reactor as a Counter-Diffusional Biofilm. Current Opinions in Biotechnology. Volume 38, April 2016, Pages 131–136. DOI:10.1016/j.copbio.2016.01.015
[3] K. Martin, Nerenberg, R. (2012). The Membrane Biofilm Reactor (MBfR) for Water and Wastewater Treatment:
Principles, Applications, and Recent Developments. Bioresource Technology. DOI: 10.1016/j.biortech.2012.02.110 [4] Vega, Marcela., et al. “Effect of chlorate addition on the performance and microbial community of a perchlorate-reducing biofilm reactor”. (submitted).
[5] Kim, B., Perez-Calleja, P., Li, M., & Nerenberg, R. (2020). Effect of predation on the mechanical properties and detachment of MABR biofilms. Water Research, 186, 116289.
[6] Aybar, M., Perez-Calleja, P., Li, M., Pavissich, J. P., & Nerenberg, R. (2019). Predation creates unique void layer in membrane-aerated biofilms. Water research, 149, 232-242.
142 Presenting Author
Bumkyu Kim
University of Notre Dame
Is the presenting author an IWA Young Water Professional? Y (i.e. an IWA member under 35 years of age)
Bio: Bumkyu Kim is a Ph.D candidate at University of Notre Dame. His research focus is on the effects of protozoa on biofilm reactors.
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