Electricity Generation From Serratia marcescens

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Gram negative bacteria like Serratia marcescens can be isolated from it and can be used to produce electricity. It was accomplished with Microbial Fuel Cells ...
Research Article

Electricity Generation From Serratia marcescens Isolated From Aerobic Sludge Using Microbial Fuel Cell Technology And Its Optimization *K. Ghanapriya1, S. Rana and P.T. Kalaichelvan1

Abstract The production of energy from wastewater is a high priority for our society given the trends of over population and worldwide energy resource depletion. Aerobic sludge from wastewater treatment plant can be used to produce electricity. MFCs can be operated using pure cultures. Gram negative bacteria like Serratia marcescens can be isolated from it and can be used to produce electricity. It was accomplished with Microbial Fuel Cells (MFCs). A microbial fuel cell (MFC) is a device that converts chemical energy into electricity through the catalytic activities of microorganisms like bacteria. Mutant strain of this bacterium produces higher amount of electricity compared to the wild strain. Although there is great potential of MFCs as an alternative energy source, extensive optimization of this bacterium has been done in this article to exploit the maximum microbial potential from it. Keywords: Serratia marcescens, Microbial Fuel Cell (MFC), electricity, mutant strain, optimization, salt bridge.

Introduction Every year the global energy demand increases. While petroleum products currently supply much of this demand, the increasing difficulty of sustained supply and the associated problems of pollution and global warming are acting as a major impetus for research into alternative renewable energy technologies. Fuel cells offer a possible (and partial) solution to this problem, with the fuel needed for conventional cells usually being either hydrogen or methanol, although some cells have been developed which run on other fuels such as hydrocarbons. A fuel cell is an electrochemical device that converts the chemical energy of a fuel (hydrogen, natural gas, methanol, gasoline, etc.) and an oxidant (air or oxygen) into electricity (Yazici, 2007). A fuel cell is comparable to an electrolytic cell or battery, where chemicals are oxidized or reduced electrochemically to produce electrcity (Smith et al., 2001). Among fuel cells, Microbial Fuel Cells (MFCs) are special types of bio-fuel cells. It is a device that converts chemical energy into electricity through the catalytic activities of microorganisms (Allen and Bennetto, 1993). Microbial fuel cells seek to add the diversity of microbial catalytic abilities to this high-efficiency design, allowing organic compounds, from simple carbohydrates to waste organic matter, to be converted into electricity (Wingard et al., 1982). They are an alternative to conventional methods of generating electricity, for small scale applications (Bennetto, 1990). Microbial fuel cells have potential to generate electricity from a wide variety of organic wastes while oxidising the wastes to less harmful

forms (Moon et al., 2006; Ieropoulos et al., 2005; Liu et al., 2004). Research into developing efficient MFC's remains a very current field, with both engineering and biological challenges yet to be met (Moon et al., 2006; Mohan et al., 2007). Microbial fuel cells have also been developed from bacteria like Bacillus spp. (Choi et al., 2004), Escherichia coli (Zhang et al., 2006), Pseudomonas aeruginosa (Rabaey et al., 2005b), Serratia marcescens (Kim and Kwon, 1999; Chang et al., 2003) etc. In this article, we would see the maximum generation of electricity from Serratia marcescens isolated from mixed culture of aerobic sludge from wastewater treatment plant by optimizing with various nutrient sources and mediators both in wild and mutant strains of the microorganism. Microbial fuel cell usually comprise of four major components anode compartment, cathode compartment, ion exchange membrane and the electrodes. Anode compartment forms the biological compartment of MFC, as it consists of microbes (biocatalyst) either in pure/mixed form (Bond and Lovley, 2003, 2005; Chaudhuri and Lovley, 2003; Holmes et al., 2004; Kim et al., 1999), which oxidizes the organic substances (fuel such as carbohydrates (Allen and Bennetto, 1993)) in the wastewater and releases free electrons. The bacterial growth in this chamber produces the necessary protons and electrons through metabolic reactions. The metabolic reactions are not allowed to proceed to completion and the intermediate electrons are drawn from the cell to do the electrical work (Mohan et al., 2007).

1.Centre for Advanced Studies in Botany, University of Madras, Chennai-600 025, Tamil Nadu (India) 2. School of Biotechnology, Guru Ghasidas, University, Bilaspur-495 001, Chhattisgarh (India) *Corresponding Author : Email Id - [email protected]

Research Article

Cathode compartment is the abiotic compartment of MFCs where the released electrons (from anode) are transferred to oxygen as a terminal electron acceptor. The ion exchange membrane helps in the transfer of protons from the anode compartment to the cathode compartment and helps to physically block oxygen diffusion into the anode chamber (Chae et al., 2008). Hence it is generally called Proton Exchange Membrane (PEM). Commonly used Proton Exchange Membranes are Nafion or Ultrex. Since PEMs are costly, here we have used salt bridges to maintain electroneutrality and allow current to flow (Booki et al., 2005). The salt bridge contains a saturated solution of some inert salt, usually Sodium chloride, Potassium chloride (Manzoni et al., 2004), or Potassium nitrate. The salt is chosen specifically to be inert based on the rest of the reagents in the system.

Nutrient Agar medium with pH 7.2 at 4°C. This was used as the wild strain. Mutant strains were obtained by treating the culture under ultra violet light for 30 mins. The mutant strain was maintained on Nutrient Agar medium with pH 7.2 at 4°C. All the incubations were done at 28°C for 48 hrs.

The major role of electron transfer is due to electrodes. Some commonly used electrodes are carbon rod, carbon/graphite sheet (Venkatamohan et al., 2008; Pham et al., 2004), stainless steel, glassy carbon, etc. The electron transfer from microbial cells to the electrode is facilitated by mediators such as thionine, methyl viologen, methyl blue, humic acid, neutral red and so on (Delaney et al., 1984). These are mediator microbial fuel cell. A number of mediators have been suggested for use in microbial fuel cells. These include natural red, methylene blue, thionine or resorfuin (Park and Zeikus, 2000; Bennetto, et al., 1983).

Figure 2a Pictorial Representation Of Microbial Fuel Cell Set-Up Using Salt Bridge

Materials and Methodology Chemicals from Himedia Laboratories Pvt. Ltd., Mumbai, Loba Fisher Chemicals and Sisco Research Laboratories, Mumbai, RFCL Ltd., New Delhi were used. Bacterial sample

Figure 1

Bacterial culture was isolated from the aerobic sludge collected from the Sewage Treatment Plant, Koyembedu, Chennai by plating on Nutrient agar media. The deep red coloured bacterial colony as in Figure 1 was further biochemically tested and identified as Serratia marcescens as shown in Table 1. The culture was maintained on

Various Biochemical tests that were done to identify the isolated organism as Serratia marcescens Serial Number 1 2 3 4 5 6 7 8 9 10 11 Table 1

Name of the test Gram staining Catalase test Indole test Methyl red test Voges-Proskauer test Citrate utilization test Glucose fermentation test Lactose fermentation test Starch hydrolysis test Urease test Casein agar test

Result + + + + + +

Figure 2b Microbial Fuel Cell Set-Up Using Salt Bridge

MFC set-up construction: A two chambered fuel cell was constructed. Two plastic containers each with diameter 20 mm were taken and marked cathode and anode. Two holes of diameter 6 mm and 1.5 mm were made on each of the lids for the insertion of the salt bridge and electrodes as shown in Figure 2a. In the anode container, 60 mL bacterial culture was inoculated and in the cathode container 60 mL potassium permanganate solution was used and the container lids were closed and sealed with tape as shown in Figure 2b. Salt bridge preparation: Salt bridge was made with 5 mm diameter level tube. The salt bridge contained a mixture of 1M potassium chloride with 5% Agar. The mixture was sucked into the level tube. This salt bridge was inserted into both the containers through one hole on both containers and sealed with tape. Different salt bridges were prepared by replacing potassium chloride with potassium nitrate, sodium chloride and in various combinations.

Research Article Electrodes used Pencil lead with diameter 1mm and length 18mm was used as electrodes to collect the electrons in both anode and cathode with copper wire connections at the other hole on both the containers and sealed with tape. Graphite sheet of 0.5mm (thick) X 20mm (length) X 10mm (breadth), carbon rod of 2mm (diameter) X 20mm (length) were also used as electrodes. These electrodes were relatively inexpensive and available easily. The electrodes were first soaked in 100% ethanol for 30 mins. After this the electrodes were washed in 1M hydrochloric acid followed by 1M sodium hydroxide, each for 1hr to remove possible metal and inorganic contaminations and to neutralize them. They were then stored in distilled water before use.

salt bridges, various electrodes, various pH values, various mediators and various electrolytes which showed the maximum electricity generation were combined and connected in series and parallel connections to give higher electricity.

Results and discussions As cited in the literature, MFCs using pure culture could be operated (Bond and Lovley, 2003; Chaudhuri and Lovley, 2003; Holmes et al., 2004) using Serratia marcescens with salt bridge. Both the wild and mutant strains generated voltage as shown in Table 3. But as shown in Figure 3, mutant strain generated more voltage compared to the wild strain. Mutant strain reached upto a maximum of 0.61V whereas the wild strain yielded only 0.43V.

Anode chamber preparation/Bacterial inoculation: Both wild and mutant strains of the above bacterial culture was inoculated in nutrient broth of pH 7.2 and incubated at 28°C at 120 rpm in shaker for 48 hrs. sixty milli litres of the bacterial culture was inoculated in the anodic chamber. Optimization of these cultures was done for better electricity generation by preparing nutrient broth at various pH and various sources (1g each) as shown in Table 2. Further optimization was done by adding various mediators to the culture broth to facilitate the electron transfer to the electrodes. The same was carried out with the mutant strain of this bacterium. Various sources used for the optimization of the wild and mutant strains of Serratia marcescens Nutrient sources

Sources used

Carbon sources

Glucose, lactose, maltose, sucrose, fructose, arabinose, mannose

Nitrogen sources

Ammonium nitrate, Urea, Peptone, Beef extract, Yeast extract

Sulphur sources

Copper sulphate, ferrous sulphate, magnesium sulphate, potassium sulphate Dipotassium hydrogen orthophosphate, Potassium dihydrogen orthophosphate Asparagine, Glycine, Histidine, Tyrosine

Phosphorus sources Amino acid sources

Figure 3

Showing the readings given by wild and mutant strains of Serratia marcescens with Potassium permanganate as electrolyte and Pencil lead as electrode Time(hrs) 0 6 12 18 24

Wild strain (V) 0.31 0.34 0.36 0.40 0.43

Mutant strain (V) 0.52 0.55 0.58 0.59 0.61

Table 3

Table 2

Cathode chamber preparation For the cathode chamber, 0.1M potassium permanganate solution was prepared. Further, various electrolyte solutions of 0.1M Potassium dichromate and 0.1M Potassium ferricyanide were used. The voltage was checked with a Multimeter (UNITY DT-830D). Operational conditions The MFCs were operated using wild and mutant strains of Serratia marcescens culture for 24 hrs at a room temperature of 33°C. The operations were carried out with different electrodes, different electrolytes, different salt bridges, different mediators and at varying pH values. Further the strains were optimized with different sources of nitrogen, carbon, sulphur, phosphorus and amino acid for maximum generation of electricity. The specific MFCs of wild and mutant strains with various optimized sources (Carbon, Nitrogen, Sulphur, Amino acid, Phosphorus); various

Dramatic increase in the voltage output is possible through various modifications of the MFC (Park and Zeikus, 2003). There is an increase in the electrical potential generation associated with the bacterial oxidation of various sources, electrode material, mediators, catholytes used, pH variation and composition of the salt bridge. (Park and Zeikus, 2003). Showing the comparative readings given by wild and mutant strains of Serratia marcescens with Potassium permanganate and various electrodes Wild strain (V) Mutant strain (V) Time(hrs) Pencil Carbon Graphite Pencil Carbon Graphite lead rod sheet lead rod sheet 0 0.31 0.75 0.41 0.52 0.29 0.34 6 0.34 0.71 0.44 0.55 0.34 0.44 12 0.36 0.70 0.45 0.58 0.35 0.49 18 0.40 0.66 0.48 0.59 0.38 0.56 24 0.42 0.61 0.50 0.61 0.39 0.63 Table 3

Research Article When different electrodes like pencil lead, graphite sheet and carbon rod were used, as shown in Table 4, wild strain generated more voltage with pencil lead whereas mutant strain generated more voltage with Graphite sheet. With various salt bridges of potassium chloride, sodium chloride, Potassium nitrate and combination of these three, wild strain generated more voltage with Potassium nitrate as in Figure 4 and mutant strain with Potassium chloride as in Figure 5.

When various catholytes like potassium permanganate, Potassium ferricyanide and Potassium dichromate were used, it was seen that in both the wild and mutant strains, maximum voltage generation was seen with Potassium permanganate as in Figure 6 and Figure 7. MFC using permanganate generated more voltage than that produced by hexacyanoferrate (Kim et al., 2007).

Figure 8

Figure 4

Figure 9 Figure 5

Figure 10 Figure 6

Figure 7

Figure 11

Research Article showing readings given by wild strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Carbon sources Time(hrs) Glucose(V) Lactose (V) Maltose (V) Sucrose (V) Fructose (V) Arabinose (V) Mannose (V)

0 6 12 18 24

0.42 0.43 0.44 0.44 0.46

0.58 0.58 0.56 0.53 0.51

0.51 0.53 0.54 0.55 0.56

0.45 0.48 0.0 0.53 0.58

0.67 0.66 0.64 0.60 0.59

0.40 0.38 0.34 0.29 0.25

0.42 0.43 0.43 0.44 0.44

Showing readings given by mutant strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Carbon sources Time(hrs) Glucose Lactose Maltose Sucrose Fructose Arabinose Mannose 0.40V 0.42V 0.44V 0.47V 0.50V

0.37V 0.37V 0.36V 0.36V 0.35V

0.39V 0.41V 0.44V 0.46V 0.49V

0.59V 0.55V 0.48V 0.39V 0.31V

0.43V 0.41V 0.40V 0.39V 0.37V

0.33V 0.33V 0.33V 0.32V 0.32V

The MFCs were optimized with various nutrient sources. With the substrates like glucose, fructose there was an increase in the voltage (Biffinger et al., 2007). As shown in Table 5a and 5b, with different Carbon sources, maximum generation in wild strain was seen with Fructose (0.59 V) and in mutant strain with Mannose (0.50 V).

Table 5a

0 6 12 18 24

output. As shown in Figure 10 and Figure 11, in both wild and mutant strains there was a maximum generation with Gram's Safranin.

0.53V 0.53V 0.52V 0.51V 0.50V

With different Nirogen sources, highest yield in wild strain and mutant strain was seen with Yeast extract as shown in Table 6a and 6b which were 0.61V and 0.57V respectively. With various Sulphur sources, maximum generation in wild strain and mutant strain was seen with Potassium sulphate (0.49V) and (0.39V) as shown in Table 7a and 7b. As shown in Table 8a and Table 8b, with different Amino acid sources, maximum generation in wild strain was seen with Asparagine (0.64 V) and in mutant strain with Tyrosine (0.53 V).

Table 5b

Showing readings given by wild strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Nitrogen sources Time(hrs) Ammonium Urea Peptone Beef extract Yeast nitrate (V) (V) (V) (V) extract (V) 0 0.50 0.71 0.52 0.54 0.56 6 0.50 0.69 0.53 0.54 0.57 12 0.49 0.62 0.53 0.55 0.58 18 0.57 0.58 0.53 0.55 0.60 24 0.47 0.54 0.53 0.56 0.61

Showing readings given by wild strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Sulphur sources Copper Ferrous Magnesium Potassium Time(hrs) sulphate (V) sulphate (V) sulphate (V) sulphate (V)

Table 6a

Table 7a

Showing readings given by mutant strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Nitrogen sources Yeast Peptone Beef extract Time(hrs) Ammonium Urea extract (V) nitrate (V) (V) (V) (V) 0 6 12 18 24

0.58V 0.55V 0.52V 0.48V 0.43V

0.35V 0.37V 0.38V 0.40V 0.43V

0.37V 0.41V 0.45V 0.48V 0.51V

0.45V 0.46V 0.47V 0.50V 0.51V

0.45V 0.49V 0.51V 0.54V 0.57V

Table 6b

At varying pH values, wild strain showed maximum voltage generation at pH 6.0 as in Figure 8 and mutant strain showed maximum generation at pH 7.0 a in Figure 9. When the MFCs were operated with different mediators like Methyl Orange (MO), Methyl Viologen (MV), Methyl Red (MR), Methylene Blue (MyB), Bromothymol Blue (BB) and Gram's Safranin (GS) there was an increase in the voltage output compared to the normal output. The use of mediators like Methylene Blue, Methyl Red (Ieropoulos et al., 2005), Methyl Viologen (Mohan et al., 2008) increase the voltage

0 6 12 18 24

0.39 0.42 0.45 0.52 0.59

0.48 0.48 0.47 0.47 0.46

0.50 0.49 0.47 0.46 0.44

0.49 0.48 0.48 0.49 0.49

TABLE 7b showing readings given by mutant strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Sulphur sources Copper Ferrous Magnesium Potassium Time(hrs) sulphate (V) sulphate (V) sulphate (V) sulphate (V) 0 6 12 18 24

0.41 0.37 0.34 0.28 0.22

0.51 0.47 0.41 0.35 0.30

0.47 0.44 0.41 0.38 0.35

0.46 0.44 0.43 0.41 0.39

Table 7b

Showing readings given by wild strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Amino acid sources Time(hrs) Asparagine (V) Glycine (V) Histidine (V) Tyrosine (V) 0 6 12 18 24 Table 8a

0.44V 0.50V 0.53V 0.58V 0.64V

0.58V 0.57V 0.56V 0.55V 0.53V

0.49V 0.43V 0.40V 0.37V 0.29V

0.32V 0.35V 0.36V 0.38V 0.39V

Research Article

Showing readings given by mutant strain of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in various Amino acid sources Time(hrs) Asparagine (V) Glycine (V) Histidine (V) Tyrosine (V) 0 6 12 18 24

0.44 0.40 0.38 0.35 0.34

0.67 0.59 0.53 0.49 0.44

0.53 0.44 0.32 0.23 0.12

0.47 0.48 0.50 0.52 0.53

Table 8b

Showing comparative readings given by wild and mutant strains of Serratia marcescens with Pencil lead, Potassium permanganate and Potassium chloride salt bridge with an optimization in Phosphorus sources Wild strain Time(hrs)

0 6 12 18 24

Mutant strain

Dipotassium Potassium Dipotassium Potassium hydrogen dihydrogen hydrogen dihydrogen orthophosphate (V) orthophosphate (V) orthophosphate (V) orthophosphate (V)

0.47 0.45 0.42 0.40 0.38

0.49 0.49 0.47 0.46 0.46

0.11 0.31 0.45 0.52 0.59

0.73 0.72 0.70 0.68 0.67

Table 9

Showing the readings given by wild strain of Serratia marcescens with Graphite sheet, Potassium permanganate, Potassium nitrate salt bridge and Asparagine with Gram's Safranin as the mediator at pH 6.0 in Series and Parallel connections. Time(hrs) Series connection (V) Parallel connection (V) 0 6 12 18 24

2.11 2.17 2.20 2.14 2.10

0.41 0.43 0.40 0.31 0.24

Table 10a

TABLE 10b showing the readings given by mutant strain of Serratia marcescens with Pencil lead, Potassium permanganate, Potassium chloride salt bridge and Dipotassium hydrogen orthophosphate with Gram's Safranin as the mediator at pH 7.0 in Series and Parallel connections. Time(hrs) Series connection (V) Parallel connection (V) 0 6 12 18 24

2.85 2.71 2.52 2.59 2.61

0.48 0.52 0.55 0.59 0.53

Table 10b

With various Phosphorus sources, maximum generation in wild strain was seen with Potassium dihydrogen phosphate (0.46 V) and in mutant strain with Dipotassium hydrogen phosphate (0.59 V) as shown in Table 9.

Figure 12

Now when several MFCs were connected with each other in Series and Parallel connections, it was seen that, maximum voltage generation was seen in Series connections of both the wild (2.10V ) and mutant strains (2.61V) of Serratia marcescens as shown in Table 10a and 10b. This was quite higher when compared to the Parallel connection output in both the strains as shown in Figure 12. Connecting several stacked MFCs in a series enabled the production of increased voltages (2.02 V in series) (Kim et al., 2007). Salt bridge MFC is the simplest biological fuel cell that can be designed and studied (Mohan et al., 2008).

Conclusion MFCs can be operated using the gram negative bacteria Serratia marcescens. Both the wild and mutant strain yield voltage, but the yield by the MFC using mutant strain in quite higher than that of the wild strain. The idea of using Salt bridge instead of the Proton exchange membrane is more economic as it is cost effective and easily available. Microbial fuel cells with wild strains of Serratia marcescens generated maximum voltage with graphite sheet as electrode, Potassium permanganate as catholyte, Potassium nitrate salt bridge, when optimized with Asparagine, with Gram's Safranin as the mediator and at pH 6.0. Similarly, Microbial fuel cells with mutant strains of Serratia marcescens showed maximum voltage generation with pencil lead as electrode, potassium permanganate as catholyte, potassium chloride salt bridge, when optimized with Dipotassium hydrogen phosphate, with Gram's Safranin as the mediator and at pH 7.0. Moreover, the MFCs in series connections yielded more voltage generation than the parallel connections. Such MFCs with maximum voltage generation have great application in biosensors.

Acknowledgement The authors sincerely thank Dr. Rengasamy, Director, Centre for Advanced Studies in Botany, University of Madras, for providing necessary laboratory facilities.

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