Team:Nanjing NFLS/Design
intracellular electron generation, electron across the bacterial surface, extracellular electron transfer to anode. We narrow down the improvement of the three process into the overexpression of three genes: nadE (NAD synthase gene), rhlA (rhamnosyl transferase gene), and phzM (methyltransferase encoding gene) in Pseudomonas aeruginosa. We plan to conduct the overexpression of the three genes at the same time, three plasmids together function in our bacteria. Now, we only demonstrate the overexpression respectively, due to limit time and resources. We also research for improvement in anode and cathode (chamber) of MFCs, looking for ways to modify MFCs ability in electricity producing and waste water treatment, besides synthetic biology ways. Besides an opportunistic pathogen that causes disease in plants and animals, including humans, Pseudomonas aeruginosa is also an electrochemically active bacteria (EAB), which are able to release electrons from inside their cells to an electrode in MFCs. We can harness many characteristics that P. aeruginosa evolved to help use harvest more electricity from the MFCs. We choose it for its good adaptation, less difficulty to observe, and large number of previous researches. When a planktonic P. aeruginosa attaches to a surface, it becomes sessile. If the cell multiplies and secretes a polysaccharide matrix on the surface, it forms a structure on the electrode, biofilm. Biofilms have many advantages including increase of resistance to antimicrobial agents and the ability of microbes to cooperate for nutrients and/or substrates.[1] In the case of anode respiring biofilms, i.e. biofilms transferring electrons to the conductive material, the terminal electron acceptor is not chemical but physical anode. Electricity-producing microorganisms can oxidize and degrade organic substrates to generate electrons, and transfer the electrons to the electron acceptor (anode) through the electron transport chain, thereby generating current. Nicotinamide adenine dinucleotide (NAD+) and its reduced form NADH are essential cofactors and electron carriers that are primarily involved with cellular metabolic reactions and energy production. [2] Most of the bacteria are able to convert chemical energy to intracellular electrons, but only electrochemically active bacteria (EAB) have the specific pathways to transfer intracellular electrons to extracellular electrode. Thus, electron transfer between bacteria and electrode which termed as extracellular electron transfer (EET) is the unique characteristic of EAB, and EET efficiency is one of the most important topics in the field of MFCs.[4] P. Aeruginosa are classified into the indirect type, producing an endogenous mediator such as Pyocyanin (PYO) and/or phenazine-1-carboxamide (PCA), reduced and oxidized in cycle to carry electrons. Besides act as an electron shuttle, PYO effects a wide range of cellular pathways, including biofilm formation on the anode, a key factor of electricity generation .[5] Surfactants are amphipathic compounds with both hydrophilic and hydrophobic moieties that preferentially partition at the interface between different phases; gas, liquid and solid. Biosurfactants are active compounds that are produced at the microbial cell surface or excreted, and also reduce surface and interfacial tension. Microbial surfactants offer several advantages over synthetic ones, such as low toxicity and high biodegradability, and remain active at extreme pH and salinity. [8] In particularly, exogenously addition of surfactants (such as EDTA, polyethyleneimine, tween-80) was proved to be an efficient strategy used to promote electricity generation from MFC. [9]However, chemical surfactant was usually toxic to bacteria, deter the biofilm formation as well as reduce bacteria viability, while biosurfactant (rhamnolipids and sophorolipid) showed less toxicity and had been applied to MFC. [10] Initially, we consider exogenously addition of rhamnolipids, naturally produced by P. aeruginosa. However, this can be too expensive and inapplicable to practice. More importantly, the response of bacteria to biosurfactant is largely depended on the physiology of the bacteria. The effects of biosurfactant on biofilm formation are varied at different growth phases, e.g., low concentration of rhamnolipids induced initial bacterial adherence but high concentration inhibited, while high concentration of rhamnolipids is indispensable to maintain the water channel of the mature biofilm [11]. So we choose to overexpress rhamnosyltransferase gene rhlA, a key gene for rhamnolipids synthesis, (synthesis pathway below) hoping the organism can produce more rhamnolipids to increase the power output, meanwhile can tune the gene regulation network by themselves. Fenton oxidation is one advanced oxidation process and widely applied for treating wastewater containing recalcitrant organic pollutants. We got the idea of integrating the Fenton process with Microbial Fuel Cells[12], when we helped search for cellulose pretreatment for our partner team NFLS. Protons and electrons can be acquired from the electrochemically active bacteria (EAB) in the anode of MFCs, while Fe2+ can be distributed evenly on the cathode, driving the reactions to produce Fenton reagent. The problem is an effective Fenton process needs high current density from the anode[14], which means that will consume part of the electricity output that we hope to increase by overexpression. Metal ions such as Fe3+, Ca2+, and Na+ were explored to stimulate energy output of MFCs. However, heavy metal ions have complicated effects on the bacteria in anode: they usually detrimental to bacteria at high concentration, while improve some cellular metabolism, electron shuttle production, anodic bacterial attachment at low concentration (at the level of μg/L).[17] Further research on the Pyocyanin (PYO) production in P. aeruginosa, we find oxygen plays a key role in converting 5- methyl-phenazine-1-carboxylic acid (5-MCA) into PYO, which is catalyzed by the O2-dependent monooxygenase PhzS. O2 can also regulate the quorum sensing system of P. aeruginosa and positively regulate the PYO production.[18] However, O2 also act as a electron acceptor, and interfere the transition of electron to anode, thus, decreasing the electricity output.[19] So we hope to use an aerobic and anaerobic strategy: infuse and stir oxygen during the formation of biofilm on the anode until they reach threshold cell density, while seal the MFCs and maintain anaerobic when later used for electricity production. Here, it is speculated that the strategy will induce an increased PYO production and anode biofilm formation, thus might finally enhance the electricity output of MFC. [1] Simões, S.M., Blankenship, J.T., Weitz, O., Farrell, D.L., Tamada, M., Fernandez-Gonzalez, R., Zallen, J.A. (2010). Rho-Kinase Directs Bazooka/Par-3 Planar Polarity during Drosophila Axis Elongation. Dev. Cell 19(3): 377--388.
Overview
From electron to anode, and to produce electricity, there must be three procedures:
- Pseudomonas aeruginosa
- biofilm
Focus 1: intracellular electron generation
- electron generation in microorganisms
The process by which electricity-producing microorganisms produce electrons generally includes two pathways: succinate oxidation and NADH oxidation process, while the latter one is the main pathway.
- Our focus: nadE (NAD synthase gene in P. aeruginosa)
De novo synthesis of NAD is common in organisms: the synthesis initiate from aspartic acid in most plants and some prokaryotes, and from tryptophan in most animals and eukaryotes. (synthesis pathway below) NAD synthetase, encoded by the nadE gene, catalyzes the final step in de novo synthesis and a salvage pathway for NAD biosynthesis[3]. The NAD+ level increases with nadE overexpression, thereby up-regulating genes whose products catalyze NADH synthesis (as the carrier of electrons). One hypothesis is that nadE overexpression can increase the NAD (H/+) pool at the source and increases the NADH/ NAD+ ratio, thus changing the electricity power output in EAB. Therefore, we choose to overexpress the nadE gene in P. aeruginosa.
Focus 2: extracellular electron transfer to anode
- transfer mechanisms
On the basis of their electron transfer mechanisms, they are basically classified into two types, indirect and direct. In indirect MFCs, microbes require some form of mediator for electron transfer to anode which can be obtained from outside (e.g. E. coli, Bacillus sp. etc.) or can be produced endogenously (e.g. Pseudomonas, Lactobacillus etc.). On the other hand, in direct MFCs bacteria known as electroactive bacteria (e.g. Shewanella putrefaciences and Geobacter sulferreducens) donate electrons directly to anode, through outer membrane redox active proteins (c-type cytochromes) or conductive nano-wire. They are also known as anodophiles or exoelectrogens and are able to form conductive biofilm on anode.
- Our focus: phzM (methyltransferase encoding gene in P. aeruginosa)
PYO is produced from chorismic acid via the phenazine pathway. (synthesis pathway below) P. aeruginosa PAO1 contains a complex phenazine biosynthetic pathway consisting of two seven-gene homologous operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, responsible for synthesis of phenazine1-carboxylic acid (PCA) [6] Subsequent conversion of PCA to PYO is mediated by two phenazine-modifying genes, phzM and phzS [7] We choose to overexpress phzM to enhance the electricity generation.
Focus 3: electron across the bacterial surface
- biosurfactant on the cell membrane
- Our focus: rhlA (rhamnosyl transferase gene in P. aeruginosa)
Focus 4: cathode
Fe-Mn/GF composite cathode
In Fenton process, the mixture of Fe2+ and H2O2 produces hydroxide (·OH) to oxidize pollutants finally into harmless products. While H2O2 can be quickly decomposed, we prefer to use in situ generation of H2O2[13] by Electron-Fenton process below:
H2O2 + Fe2+ à Fe3+ + ·OH + OH-
To improve the Fenton catalytic activity under limited current density, we decided to develop a Bio-Electron Fenton system equipped with a novel Fe-Mn/GF composite cathode[15], without using expensive noble metals. Recent studies demonstrated that introduction of Mn into Fe to form Fe-Mn binary oxides exhibits excellent activity for the degradation of contaminants. For instance, Fe-species-loaded mesoporous manganese dioxide (Fe/M-MnO2) was fabricated by a complex method, which shows higher efficiency and more remarkable stability than mesoporous manganese dioxide (M-MnO2) for discoloration of methylene blue.[16] We hope the cathode covered with a combination of Fe and Mn will also improve the waste water treating ability of MFCs.
Focus 5: anode
- metal ions concentration
We consider testing copper (II) (CuSO4), cadmium (II) (CdCl2), zinc (II) (ZnCl2), chromium (III) (CrCl3), manganese (II) (MnCl2), cobalt (II) (CoCl2), nickel (II) (NiSO4) in future experiment. After interviewing with experts in companies (view in Human Practices), we also intend to use low concentration waste water from satisfactory resources, to reduce the cost of energy source.
- aerobic and anaerobic strategy
view kill switch for safety and hardware operation in Implementation page.
[2]Förster J, et al. (2003) Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network. Genome Res 13(2):244-53
[3] Lai, B., Tang, X., Li, H., Du, Z., Liu, X., Zhang, Q., 2011. Biosens. Bioelectron. 28, 373–377.
[4] Logan, B.E., 2009. Exoelectrogenic bacteria that power microbial fuel cells. Nat. Rev. Microbiol. 7, 375–381.
[5] Dietrich, L.E.P., Okegbe, C., Price-Whelan, A., Sakhtah, H., Hunter, R.C., Newman, D. K., 2013. Bacterial community morphogenesis is intimately linked to the intracellular redox state. J. Bacteriol. 195, 1371–1380.
[6] Yong, Y.C., Zhong, J.J., 2013. Impacts of quorum sensing on microbial metabolism and human health. Adv. Biochem. Eng. Biotechnol. 131, 25–61.
[7] Greenhagen, B.T., Shi, K., Robinson, H., Gamage, S., Bera, A.K., Ladner, J.E., Parsons, J.F., 2008. Crystal structure of the pyocyanin biosynthetic protein PhzS. Biochemistry 47, 5281–5289.
[8] Kapadia, S.G. and Yagnik, B.N. (2013) Current Trend and Potential for Microbial Biosurfactants. Asian Journal of Experimental Biological Sciences, 4, 1-8.
[9] Liu, J., Qiao, Y., Lu, Z.S., Song, H., Li, C.M., 2012. Enhance electron transfer and performance of microbial fuel cells by perforating the cell membrane. Electrochem. Commun. 15, 50–53
[10]Shen, H.B., Yong, X.Y., Chen, Y.L., Liao, Z.H., Si, R.W., Zhou, J., Wang, S.Y., Yong, Y.C., OuYang, P.K., Zheng, T., 2014. Enhanced bioelectricity generation by improving pyocyanin production and membrane permeability through sophorolipid addition in Pseudomonas aeruginosa-inoculated microbial fuel cells. Bioresour. Technol. 167, 490–494.
[11]Nickzad, A. and Déziel, E. (2014), The involvement of rhamnolipids in microbial cell adhesion and biofilm development – an approach for control?. Lett Appl Microbiol, 58: 447-453. doi:10.1111/lam.12211
[12] Babuponnusami, A., Muthukumar, K., 2014. A review on Fenton and improvements to the Fenton process for wastewater treatment. J. Environ. Chem. Eng. 2, 557---572.
[13] Ling, T., Huang, B., Zhao, M., Yan, Q., Shen, W., 2016. Repeated oxidative degradation of methyl orange through bio-electro-Fenton in bioelectrochemical system (BES). Bioresour. Technol. 203, 89--95.
[14] Xu, N., Zeng, Y., Li, J., Zhang, Y., Sun, W., 2015. Removal of 17b-estrodial in a bioelectro-Fenton system: contribution of oxidation and generation of hydroxyl radicals with the Fenton reaction and carbon felt cathode. RSC Adv. 5, 56832-56840.
[15] Zhang, L., Yin, X., Li, S.F.Y., 2015. Bio-electrochemical degradation of paracetamol in a microbial fuel cell-Fenton system. Chem. Eng. J. 276, 185--192.
[16] Huang, G., Wang, H., Zhao, H., Wu, P., Yan, Q., 2018. Application of polypyrrole modified cathode in bio-electro-Fenton coupled with microbial desalination cell (MDC) for enhanced degradation of methylene blue. J. Power Sources 400, 350--359.
[17] Paraszkiewicz, K., Frycie, A., Slaba, M., Dlugonski, J., 2007. Enhancement of emulsifier production by Curvularia lunata in cadmium, zinc and lead presence. Biometals 20, 797–805.
[18] Recinos, D.A., Sekedat, M.D., Hernandez, A., Cohen, T.S., Sakhtah, H., Prince, A.S., Price-Whelan, A., Dietrich, L.E.P., 2012. Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and different roles in pathogenicity. 19420-19425
[19] Cheng, H.Y., Liang, B., Mu, Y., Cui, M.H., Li, K., Wu, W.M., Wang, A.J., 2015. Stimulation of oxygen to bioanode for energy recovery from recalcitrant organic matter aniline in microbial fuel cells (MFCs). Water Res. 81, 72–83.