Investigating Ammonia-producing Bacteria for the Establishment of a ph Gradient in Microbial Biofuel Cells Alex Hodson Mentor: Dr. R. Shane Gold BIOL 491-494 April 20 2015
Abstract Microbial biofuel cells (MFCs) present a unique opportunity to recycle organic waste, such as old food or yard waste, into electricity by harnessing the metabolic activities of bacteria. Traditionally MFCs have utilized two chambers filled with bacteria growing at a neutral ph to produce an open circuit voltage potential; power production using this design has been moderately successful. In a recent modification, a manually generated ph gradient established between the two chambers demonstrated improved power output, but the need to maintain the gradient using exogenous HCl and NaOH has proven to be expensive. This research investigates the use of ammonia-producing bacteria for potential use in the cathodic chamber to help establish and maintain a natural ph gradient without the need for exogenous ph adjustment. Growth curves, ph levels, and ammonium ion concentrations were measured for Azotobacter vinelandii and Proteus mirabilis grown with and without urea. The alkalinization coefficient (Ealk), defined as the average change in ph divided by the average change in growth, was used to compare the potential of bacteria to establish and maintain a ph graident within a MFC. Proteus mirabilis yielded an Ealk of 3.1667 and 2.7354 when grown with, and without, urea, respectively; Azotobacter vinelandii yielded an Ealk of 0. These results suggest that of these two species, P. mirabilis shows the greatest potential at raising the ph for increased energy production in the cathodic chamber of a gradient based MFC. Keywords: Microbial fuel cell, Proteus mirabilis, Azotobacter vinelandii, ph gradient, organic mediator dyes, Lactobacillus acidophilis Introduction The microbial fuel cell (MFC) represents a novel technology to simultaneously generate electric power and treat wastewater using both pure organic matter and wastewater as fuel to generate electric power (Wang et al. 2014). MFCs use the metabolism of heterotrophic bacteria to generate protons and electrons from an organic fuel source. As protons are generated they are able to pass through a proton exchange membrane from the anode to the cathode while electrons are forced to pass through an external wire, thereby producing an electric current (Rahimnejad et al. 2011). In the course of MFC operation the protonaccepting side (cathode) naturally becomes acidified as protons accumulate which 1
results in a reduction in power output (Zhuang et al. 2010). Zhuang et al. (2010) added HCl and NaOH continuously to the anode and cathode sides, respectively, to maintain a cathodic ph of 10.0 and an anodic ph of 2.0; this system was observed to achieve open circuit voltages and maximum power densities 1.5 and 3.8 times higher than those obtained in the same MFC working at a neutral ph. While voltage and power density increased by maintaining a ph gradient using exogenous agents, the constant addition of HCl and NaOH is costly and inefficient. The use of acidproducing bacteria in the anode and alkali-producing bacteria in the cathode may help to establish and maintain the ph gradient required to optimize energy production spontaneously without the need for supplementation. A number of bacteria are able to create an alkaline environment through the production of ammonia by hydrolyzing urea, hydrolyzing proteins, or through nitrogen fixation (Vince et al. 1973, Padan et al. 2005, Thomspson and Zher 2013). The purpose of this study was to investigate the use of alkali-producing bacteria for potential use in the cathode chamber side of a gradient based MFC. Methods Azotobacter vinelandii (ATCC #478), Proteus mirabilis (ATCC # 25933), and Lactobacillus acidophilus (ATCC #4356) were obtained from the American Type Culture Collection. Azotobacter vinelandii was cultured on Azotobacter medium (1.5 mm KH2PO4, 4.6 mm K2HPO4, 0.8 mm MgSO4 x 7H2O, 0.6 mm CaSO4 x 2H2O, 0.06 mm FeCL3, 0.05 mm Na2MoO4 x 2H2O, and 0.05 g yeast extract per liter), Proteus 2
mirabilis was cultured in Nutrient Broth (Difco), and L. acidophilus was cultured in MRS broth (Difco), each at 37 0 C with vigorous shaking (250 rpm). To determine the ability of A. vinelandii and P. mirabilis to establish an alkaline environment the optical density (OD 600 nm) and ph were monitored by taking samples hourly for 21-36 hours. Each hourly sample was assayed for ammonium ion concentration by acidifying to ph 4.0 with acetate buffer and tested with an ammonium ion-selective electrode (Vernier). To determine the effects of bacterial strains on current, voltage, and power output a microbial biofuel cell was constructed of two 250 ml Wheaten Bottles with a 5.5 mm diameter proton selective membrane used to separate the anodic and cathodic chambers. A titanium electrode (#313830, Millrose) integrated with a carbon fiber brush (VWR, 2.5 cm X 2.5 cm X 7.2 cm) was used to facilitate the movement of the electrons to the external circuit. Lactobacillus acidophilus was inoculated into the anodic chamber and A. vinelandii or P. mirabilis was inoculated into appropriate media within the cathodic chamber. The MRS broth in the anode was supplemented with 20 mm indigo carmine to serve as the mediator for electron transfer. Electrical output and voltage were measured continuously for 24 hours. The tests were performed in triplicate and significance was analyzed using ANOVA. Results The slope of the logarithmic (linear) phase of growth and ph were calculated as a means of comparing growth rates and ph change between bacterial species (Figures 1 and 2). The alkalinization coefficient (Ealk) was determined by dividing 3
Optical density the average change in ph by the average change in optical density. Proteus mirabilis yielded an Ealk of 2.7 and 3.2 when grown with, and without, urea, respectivly (Figure 3); Azotobacter vinelandii yielded an Ealk of 0. Figure 1: The fuel cell setup used in this study. 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 25 Time (h) Azotobacter vinelandii Proteus mirabilis (no urea) Proteus mirabilis (urea) Figure 2. Optical density measured over 24 hours. 4
Ammonium mg/l ph 9 8.5 8 Azotobacter vinelandii 7.5 Proteus mirabilis (no urea) 7 Proteus mirabilis (urea) 6.5 0 5 10 15 20 Time (h) Figure 2. ph measured over 16 hours. 25 20 15 Azotobacter vinelandii 10 5 Proteus mirabilis (no urea) Proteus mirabilis (urea) 0 0 5 10 15 20 25 Time (h) Figure 3. Ammonium ion concentration measured over 24 hours. Discussion Microbial bio fuel cells hold great potential for the production of electricity from biological waste streams. If effectively optimized, MFCs could be used to power cities from sewage, as power sources for pacemakers that do not require battery 5
replacement, or as home power systems that run from organic material such as food scraps or yard waste. The establishment of a natural ph gradient may help improve the efficiency of MFC s to make this technology competitive with current energy production techniques. The bacteria that are able to produce the greatest increase in ph, thereby establishing the greatest ph gradient at the lowest metabolic rate, may be most ideal for use in the cathodic chamber. Of the strains tested, P. mirabilis, with an alkalinization coefficient of 3.2, increased the ph to the greatest extent while maintaining a low metabolic rate. When P. mirabilis was tested with urea the metabolic rate increased without a corresponding increase in ph, making these conditions less suitable for MFC use. Ammonium ion concentrations were measured to identify the metabolic process by which ph is increased; however, no correlation was observed between an increase in ammonium ion concentration and a raise in ph. The ability of P. mirabilis to rapidly raise the ph of the cathodic environment confers the potential to neutralize protons passing through the proton-selective membrane from the anodic chamber, thereby establishing and maintaining a ph gradient that has the potential to maximize power output without the use of exogenous agents. These results indicate that further studies involving P. mirabilis in a microbial fuel cell, in conjunction with an acid-producing anodic organism, are warranted in an effort to improve the efficiency and cost effectiveness of the microbial fuel cell technology. References Padan, E., E. Bibi, M. Ito and T. Krulwich. 2005. Alkaline ph Homeostasis in Bacteria: New Insights. Biochim Biophys Acta 1717(2): 67-88. 6
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