Article pubs.acs.org/est
Fermentation of Glycerol into Ethanol in a Microbial Electrolysis Cell Driven by a Customized Consortium Allison M. Speers,† Jenna M. Young,†,‡ and Gemma Reguera* Department of Microbiology and Molecular Genetics, Michigan State University, 6190 Biomedical and Physical Science Building, 567 Wilson Road, East Lansing, Michigan 48824, United States S Supporting Information *
ABSTRACT: The in situ generation of ethanol from glycerol-containing wastewater shows promise to improve the economics of the biodiesel industry. Consequently, we developed a microbial electrolysis cell (MEC) driven by the synergistic metabolisms of the exoelectrogen Geobacter sulfurreducens and the bacterium Clostridium cellobioparum, which fermented glycerol into ethanol in high yields (90%) and produced fermentative byproducts that served as electron donors for G. sulfurreducens. Syntrophic cooperation stimulated glycerol consumption, ethanol production, and the conversion of fermentation byproducts into cathodic H2 in the MEC. The platform was further improved by adaptively evolving glycerol-tolerant strains with robust growth at glycerol loadings typical of biodiesel wastewater and by increasing the buffering capacity of the anode medium. This resulted in additional increases in glycerol consumption (up to 50 g/L) and ethanol production (up to 10 g/L) at rates that greatly exceeded the capacity of the anode biofilms to concomitantly remove the fermentation byproducts. As a result, 1,3-propanediol was generated as a metabolic sink for electrons not converted into electricity syntrophically. The results highlight the potential of consortia to process glycerol in MECs and provide insights into genetic engineering and system design approaches that can be implemented to further improve MEC performance to satisfy industrial needs.
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INTRODUCTION The increased concern for the inevitable depletion of the oil supply as well as the negative impact of the use of fossil fuels on the environment has highlighted the need for biofuel alternatives such as ethanol, diesel, butanol, hydrogen, and electricity produced from renewable plant biomass.1 Biodiesel is a promising transportation fuel alternative because it is chemically analogous to petrochemical diesel, which fuels compression engines, and can be distributed using the existing infrastructure.2 Furthermore, it can be produced from dedicated agricultural oil feedstocks such as soybeans with relatively low inputs and/or minimum impacts on existing agricultural practices, rural economies, and the environment.3 The economic and environmental viability of the biodiesel industry is, however, limited by the large volumes of glycerol-containing wastewaters generated during production, which most often require disposal for a fee at water treatment facilities.4,5 Wastewater with approximately 40−50% glycerol is generated after the phase separation of the crude biodiesel, but the glycerol is further diluted to ∼10% after the addition of wastewater generated from washing of the crude biodiesel.4,5 Glycerol prices were traditionally high enough to allow producers to generate profit from refining the diluted glycerol waste, concentrating it to an 80% stock, and selling it to glycerol biorefineries.4 However, the rapid growth of the biodiesel industry in the last two decades has produced glycerol in excess of its demand, which has dropped prices dramatically.6 Furthermore, bioethanol production also generates glycerol byproduct up to 10% (w/w) of the total sugar consumed.7 In © XXXX American Chemical Society
this saturated market, glycerol has become a very low value or waste product for biodiesel producers, and glycerol-containing wastewaters are often an economic and environmental liability to the biodiesel industry. Technologies are being explored to use the refined glycerol as a boiler fuel or as a substrate for the generation of commodity chemicals, fertilizers, and extenders in animal feeds.6 However, there is growing interest in developing technologies that would allow producers to directly process the glycerol-containing wastewater into value-added products, thereby bypassing the need to pretreat the water streams to remove impurities and concentrate the glycerol. The reduced nature of glycerol compared with sugars also makes it an attractive substrate for microbial fermentations and for the production of commodity and/or specialty chemicals.8,9 For example, glycerol reduction can be used to generate 1,3propanediol, a valuable precursor for a formulation of polyester (polypropylene terephthalate) and for the synthesis of biodegradable plastics.8,10 The fermentative production of ethanol from glycerol has also been explored.11 This approach is particularly attractive to improve the economics of the biodiesel industry because it could be performed onsite to generate an alcohol feedstock for the transesterification reaction of triacylglycerides, which produces biodiesel and glycerol in the presence of a salt catalyst.12 Methanol derived from natural Received: February 10, 2014 Revised: April 30, 2014 Accepted: May 6, 2014
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dx.doi.org/10.1021/es500690a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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well as optimization of the growth medium resulted in a robust MEC platform that stimulated glycerol consumption and ethanol production. The studies also identified bottlenecks of the platform, which could be removed with targeted genetic engineering approaches and changes in system design to further increase MEC performance.
gas is the least expensive of all the alcohols in most countries and therefore is used more often for transesterification.2,13 However, the low production cost of bioethanol from the glycerol waste could make the biodiesel industry fully independent of fossil feedstocks while reducing costs associated with the disposal and/or treatment of the glycerin wastewater. However, despite their promise, glycerol fermentation technologies are not robust enough to meet industrial standards. The microbial fermentation of glycerol generates byproducts that reduce the molar yields of the value-added products and accumulate in the fermentation broth, slowing down or even inhibiting the growth of the fermentative organism.10 Even after extensive genetic engineering and adaptive evolution of some of the most promising fermentative strains, waste products are still generated and the fermentation efficiency is not high enough to satisfy industrial needs.6,9,10,14 Bioelectrochemical systems such as microbial fuel cells (MFCs) and microbial electrolysis cells (MECs) have also been explored for the conversion of glycerol into electricity and cathodic H2, respectively, yet their glycerol loadings and/or coulombic efficiencies are too low to make these systems commercially attractive.15−17 Genetic engineering and adaptive evolution approaches have also been used to confer on exoelectrogenic strains the ability to metabolize glycerol in bioelectrochemical systems. For example, an adaptively evolved strain of Geobacter sulfurreducens was isolated and used to generate electricity from the oxidation of glycerol in a MFC, although the substrate concentration was too low (0.05% w/v) to make this approach practical.18 In another study, the exoelectrogen Shewanella oneidensis was genetically engineered for ethanol production from glycerol, and the electrodedependent conversion of glycerol to ethanol and acetate was demonstrated in an electrochemical reactor.19 Naturally established microbial communities that partially recover energy from glycerol-containing wastewater in bioelectrochemical systems have also been reported.20 This platform is attractive because it can process the glycerol directly from the wastewater. However, the uncontrolled growth of fastidious organisms in naturally established consortia diverts electrons from glycerol, and as a result, the power densities are low.21 An alternative approach is to use laboratory microbial consortia tailored to maximize the conversion of glycerol into a value-added product such as ethanol with the simultaneous removal of waste products by an exoelectrogen, which converts them into electricity and/or cathodic H2. Such a targeted approach enabled, for example, substantial energy recoveries from chemically pretreated corn stover as ethanol and cathodic H2 in a MEC.22 The applied potential promoted the growth of exoelectrogenic biofilms on the anode electrode and maximized the conversion of fermentation byproducts into cathodic H2. In doing so, the exoelectrogenic biofilms also prevented the accumulation of feedback inhibitors and stabilized the pH of the broth, stimulating the growth of the fermentative partner and maximizing energy recoveries from fermentation alone.22 Hence, we explored the suitability of MECs driven by customized consortia to ferment glycerol when provided at loadings typical of glycerin wastewater streams (approximately 10%). The consortium included Clostridium cellobioparum, a glycerol-fermenting bacterium selected for its superior ethanologenesis from glycerol, and the exoelectrogen G. sulfurreducens, which converted waste byproducts of glycerol fermentation into electricity. Optimization of the glycerol tolerance of the microbial catalysts via adaptive evolution as
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MATERIALS AND METHODS Bacterial Strains and Culture Conditions. Fermentative strains from our culture collection [Table S1 in the Supporting Information (SI)] were grown anaerobically in GS2 medium23 supplemented with 0.2% (w/v) cellobiose (GS2−CB) before inoculation to an initial optical density at 660 nm (OD660) of 0.04 into triplicate tubes with 10 mL of GS2 containing 0.3% (w/v) glycerol (GS2−glycerol) to screen for their ability to ferment glycerol. All of the incubations for the initial screening were at 35 °C, and growth was monitored spectrophotometrically (OD660) every 12 h. C. cellobioparum (herein designated Ccel) was routinely grown at 35 °C in anaerobic GS2−CB or in GS2−glycerol medium in which glycerol was provided at various concentrations, as indicated. G. sulfurreducens PCA (herein designated Gsul) was routinely cultured at 30 °C in anaerobic DB medium24 with 20 mM acetate and 40 mM fumarate (DB−AF). When indicated, the glycerol-tolerant strains CcelA and GsulA, which were adaptively evolved from the ancestor Ccel and Gsul strains, respectively, as described in the SI, were used. For the coculture experiments, late-exponential-phase cultures of the ancestor or adaptively evolved strains of Gsul and Ccel were grown anaerobically at 30 °C in DB−AF and GS2−CB medium, respectively, and inoculated to an initial OD 660 of 0.02 in the same (coculture) or separate (monoculture) tubes containing 10 mL of GS2 medium supplemented with glycerol in various concentrations, as indicated, and 40 mM fumarate. Control monocultures for each strain were also prepared in GS2 medium without glycerol to account for any growth from the yeast extract present in the medium or from nutrients carried over in the inoculum. All of the cultures were incubated at 30 °C, and growth was monitored spectrophotometrically (OD660) every 6 h. Microbial Electrolysis Cells. Dual-chambered, H-type MECs were set up and sterilized by autoclaving as described previously.24 The reference electrode (3 M Ag/AgCl, Bioanalytical Systems Inc.) was sterilized in 70% ethanol for 1 min and rinsed with sterile water before being inserted into the anode chamber. Unless otherwise indicated, sterile DB medium was added to the anode (90 mL) and cathode (100 mL) chambers to grow Gsul or GsulA with 1 mM acetate (acetate-pregrown biofilms). After addition of the medium, the anode electrode was poised at 0.24 V vs Ag/AgCl with a VSP potentiostat (BioLogic), and the chambers were sparged with filter-sterilized 80:20 N2/CO2 gas to ensure anaerobiosis. Once the current stabilized, the anode chamber was inoculated with 10 mL of a suspension of Gsul or GsulA cells harvested from early-stationary-phase DB−AF cultures, as described before.22 MEC monoculture controls with Ccel or CcelA were inoculated with 10 mL of a cell suspension from cultures grown in GS2 medium prepared without MOPS buffer (GS3 medium) and supplemented with 3.8% or 10% glycerol, respectively. All of the cultures and MECs were incubated at 30 °C. For the consortium experiments, a sequential inoculation strategy was used. Anode biofilms of Gsul or GsulA were first B
dx.doi.org/10.1021/es500690a | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
Figure 1. Syntrophic growth of G. sulfurreducens (Gsul) and C. cellobioparum (Ccel) in GS2 medium with 0.3% glycerol and 40 mM fumarate at 30 °C. (A) Growth (OD660) of the Ccel−Gsul coculture (●), monocultures of Ccel (○) and Gsul (□), and Ccel controls without glycerol (dashed line). (B) Glycerol fermentation products in the Ccel−Gsul coculture in reference to the Ccel monoculture controls with (Ccel) or without (Ccel*) glycerol. (C) Glycerol tolerance of the coculture and Ccel and Gsul monocultures; symbols are as in (A). Shown are average growth rates and standard deviations from three replicate cultures.
(Gram-positive) cells were differentially stained with the BacLight Gram Stain Kit (Invitrogen) in green and red, respectively, following the manufacturer’s recommendations. The electrodes were imaged with an FluoView FV1000 inverted microscope (Olympus, Center Valley, PA) equipped with a PLAPON 120× oil immersion objective [Olympus; numerical aperture (NA) 1.42]. The excitation wavelength was 488 nm for both dyes. The emission spectra were detected with a BA505-525 bandpass filter (SYTO 9, green) and a BA650IF long-pass filter (hexidium iodide, red). Biofilm images were collected every 0.4 μm starting with the electrode-associated layer, and the image stacks were used to generate top or side 3D image projections using the FV10-ASW 3.0 software (Olympus). Analytical Techniques. Alcohols and organic acids in culture supernatant fluids were analyzed by high-pressure liquid chromatography (HPLC) (Waters, Milford, MA) at 30 °C as previously described,25 except that the samples were filtered with 0.45 μm syringe filters (National Scientific, Rockwood, TN) prior to analysis. When indicated, the pH of the fermentation broth was measured with an Orion Aplus pH meter (Thermo Electron, Beverly, MA). Gases in the culture’s headspace were also analyzed using a Varian CP-4900 micro gas chromatograph (Agilent, Santa Clara, CA).
grown in the anode chamber with DB medium and 1 mM acetate until all of the acetate was consumed (i.e., when the current declined to
