A Simple and Low‐Cost Procedure for Growing Geobacter sulfurreducens Cell Cultures and Biofilms in Bioelectrochemical Systems

J. Patrick O'Brien1, Nikhil S. Malvankar1

1 Department of Molecular Biophysics and Biochemistry, Microbial Sciences Institute, Yale University, West Haven, Connecticut
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix A.4K
DOI:  10.1002/cpmc.20
Online Posting Date:  November, 2016
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Abstract

Anaerobic microorganisms play a central role in several environmental processes and regulate global biogeochemical cycling of nutrients and minerals. Many anaerobic microorganisms are important for the production of bioenergy and biofuels. However, the major hurdle in studying anaerobic microorganisms in the laboratory is the requirement for sophisticated and expensive gassing stations and glove boxes to create and maintain the anaerobic environment. This appendix presents a simple design for a gassing station that can be used readily by an inexperienced investigator for cultivation of anaerobic microorganisms. In addition, this appendix also details the low‐cost assembly of bioelectrochemical systems and outlines a simplified procedure for cultivating and analyzing bacterial cell cultures and biofilms that produce electric current, using Geobacter sulfurreducens as a model organism. © 2016 by John Wiley & Sons, Inc.

Keywords: biofilm; anaerobes; gassing station; bioelectrochemical systems; microbial fuel cell; Geobacter sulfurreducens

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the Simplified Gassing Station
  • Basic Protocol 2: Preparation of Anaerobic Medium Using the Gassing Station
  • Basic Protocol 3: Inoculation and Growth of Geobacter Culture
  • Basic Protocol 4: Construction of the Bioelectrochemical System (BES)
  • Basic Protocol 5: Microbial Electrolysis Cell (MEC) Operation
  • Support Protocol 1: Testing of Media
  • Support Protocol 2: Prepare Glass Syringe Filter Assembly
  • Support Protocol 3: MEC Maintenance
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of the Simplified Gassing Station

  Materials
  • Inert gas cylinder (80:20 N 2:CO 2 for this work)
  • Pressure regulator
  • Gassing station manifold (Swagelok)
  • Copper tubing, 1/8–in. O.D. (Swagelok, cat. no. CU‐T2‐S‐030‐50)
  • Rubber tubing (Cole Parmer, cat. no. 06508‐14)
  • Male luer‐lock fittings 1/16‐in. (Cole Parmer, cat. no. 41507‐26)
  • Sterile 1‐ml plastic syringe

Basic Protocol 2: Preparation of Anaerobic Medium Using the Gassing Station

  Materials
  • NBAF medium (see recipe)
  • 10‐ml pressure tubes (Chemglass, cat. no. CLS‐4209‐10) or 125‐ml serum bottles (Fisher Scientific, cat. no. 06‐406K)
  • Assembled gassing station ( protocol 1)
  • Cannulas (reusable stainless steel needles; Cadence Science, cat. no. 7937)
  • Inert gas cylinder (80:20 N 2:CO 2 for this work)
  • Blue butyl rubber stoppers (Chemglass, cat. no. CLS‐4209‐14)
  • Aluminum crimps (Fisher, cat. no. 06‐406‐15)
  • Crimper (Chemglass, cat. no. CG‐4930‐20 or equivalent)

Basic Protocol 3: Inoculation and Growth of Geobacter Culture

  Materials
  • 70% and 100% ethanol
  • 100 mM cysteine (see recipe)
  • NBAF medium (see recipe) in 10‐ml pressure tube (see protocol 2)
  • G. sulfurreducens strain PCA (ATCC #515733), frozen stock
  • Glass syringe filter (See protocol 7), autoclaved
  • Tripod stand
  • Sterile 1‐ml syringe and 25‐G needle
  • Sterile 3‐ml syringe and 25‐G needle
  • Spectrophotometer for determining OD 600
  • 125‐ml serum bottles (Fisher Scientific, cat. no. 06‐406K)
  • Additional reagents and equipment for preparing glass syringe filter ( protocol 7)

Basic Protocol 4: Construction of the Bioelectrochemical System (BES)

  Materials
  • 0.5 M HCl
  • 0.5 N NaOH
  • Double‐distilled H 2O
  • 100% and 70% ethanol
  • Inert gas cylinder (80:20 N 2:CO 2 for this work)
  • FWAF medium, anaerobic (see recipe)
  • FWNN medium, sterile, aerobic (see recipe)
  • 10% bleach
  • Items in Figure
  • Drill
  • 25‐G needle
  • Assembled gassing station ( protocol 1)
  • Autoclaved 0.2‐µm filter (Pall, cat. no. 4423)
  • Rubber tubing (Cole Parmer, item no. EW‐06508‐14)
  • 25‐G needle
  • 21‐G BD Vacutainer Eclipse Blood Collection Needle (BD, cat. no. 368607)
  • Known standard electrode
  • Vinyl tape (of type used in anaerobic glove boxes, e.g., 3 M, cat. no. 471)

Basic Protocol 5: Microbial Electrolysis Cell (MEC) Operation

  Materials
  • Serum bottle containing at least 60 ml of cells in mid‐exponential phase (OD 600 ∼0.3; see protocol 3)
  • 100% ethanol
  • FWAF medium, anaerobic (see recipe)
  • FWA medium, anaerobic (see recipe)
  • DL vitamins (see recipe)
  • DL minerals (see recipe)
  • 10% bleach
  • Assembled gassing station ( protocol 1)
  • 25‐G needles
  • 21‐G BD Vacutainer Eclipse Blood Collection Needle (BD, cat. no. 368607)
  • Thermo Cimarec Poly 15 stir plate (Thermo Scientific)
  • Glass syringe filter prepares as in protocol 7
  • Ag/AgCl reference electrode (Electrolytica, cat. no. C‐925)
  • Potentiostat (Gamry Instruments, cat. no. 992‐00088, Interface 1000E Potentiostat or equivalent)
  • Spectrophotometer for determining OD 600
  • Masterflex PharMed BPT tubing 3/16‐in. (Cole Parmer, cat. no. EW‐06508‐15)
  • Autoclaved rubber stopper (size 6.5) with two holes
  • Sparger (Ace Glass, cat. no. 6453‐109)
  • Female luer‐lock hose connector (Cole Parmer, cat. no. EW‐41507‐30)
  • Fisherbrand Instant Sealing Sterilization Pouch (Fisher, cat. no. 01‐812‐57, or equivalent)
  • 10‐liter jug (Corning, cat. no. 1395‐10L)
  • Male Luer‐lock connectors (Cole Parmer, cat. no. EW‐41507‐26)
  • Pump tubing (2‐STOP PHARMED, 1.30 mm I.D., Cole Parmer, cat. no. EW‐95713‐32)
  • Peristaltic pump (Figure 7C.1.7, item 14; Cole Parmer, cat. no. EW‐78001‐42)
  • Walk‐in culture room set to 25°C and 30% humidity for optimal growth

Support Protocol 1: Testing of Media

  Materials
  • Pressure tubes and serum bottle with NBAF or FWAF medium to be tested (see recipes and protocol 2)
  • 100 mM cysteine (“good batch”; already tested according to the steps below)
  • “Happy” wild‐type NBAF culture of Geobacter ( protocol 3; do not allow to stand in stationary phase >2 days)
  • Serum bottle with 100 mM cysteine to be tested (see recipe and protocol 2)
  • Litmus paper

Support Protocol 2: Prepare Glass Syringe Filter Assembly

  Materials
  • Glass syringe (Cadence Science, cat. no. 5024, or Fisher, cat. no. 14‐825‐3B)
  • Glass wool (Fisher, cat. no. AC38606‐0500)
  • Rubber stopper (size #1)
  • 18‐G needle
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Figures

Videos

Literature Cited

Literature Cited
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  Malvankar, N.S., Tuominen, M.T., and Lovley, D.R. 2012d. Lack of cytochrome involvement in long‐range electron transport through conductive biofilms and nanowires of Geobacter sulfurreducens. Energy Environ. Sci. 5:8651‐8659. doi: 10.1039/c2ee22330a.
  Malvankar, N.S., Lovley, D.R., Beyenal, H., and Babauta, J. 2015. Electronic conductivity in living biofilms: Physical meaning, mechanisms, and measurement methods. In Biofilms in Bioelectrochemical Systems pp. 211‐248. John Wiley & Sons, Inc.
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