Biochemical and Biophysical Research Communications 391 (2010) 1033–1038 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc An evolved Escherichia coli strain for producing hydrogen and ethanol from glycerol Hongbo Hu a,b, Thomas K. Wood a,* a Department of Chemical Engineering, 220 Jack E. Brown Building, Texas A&M University College Station, TX 77843-3122, USA b Key Laboratory of Microbial Metabolism, Ministry of Education, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China article info abstract Article history: Glycerol is an attractive feedstock for biofuels since it accumulates as a byproduct during biodiesel oper- Received 24 November 2009 ations; hence, here we consider converting glycerol to hydrogen using the formate hydrogen lyase system Available online 11 December 2009 of Escherichia coli which converts pyruvate to hydrogen. Starting with E. coli BW25113 frdC that lacks fumarate reductase (this mutation reduces repression of hydrogen synthesis during glycerol fermenta- Keywords: tion) and by using both adaptive evolution and chemical mutagenesis combined with a selection method Adaptive evolution based on increased growth on glycerol, we obtained an improved strain, HW2, that produces 20-fold Glycerol metabolism more hydrogen in glycerol medium (0.68 ± 0.16 mmol/L/h). HW2 also grows 5-fold faster (0.25 ± 0.01/h) Bacterial hydrogen production than BW25113 frdC on glycerol, so it achieves a reasonable anaerobic growth rate. Corroborating the increase in hydrogen production, glycerol dehydrogenase activity in HW2 increased 4-fold compared to BW25113 frdC. In addition, a whole-transcriptome study revealed that several pathways that would decrease hydrogen yields were repressed in HW2 (fbp, focA, and gatYZ) while a beneficial pathway which encodes enolase was induced. Ethanol production was also increased 5-fold in the evolved HW2 strain. Ó 2009 Elsevier Inc. All rights reserved. Introduction can be converted into various interesting products such as 1,3-pro- panediol, succinic acid, dihydroxyacetone, and ethanol [5]. Diminishing supplies of fossil fuels along with environmental Another possible route is conversion of glycerol to hydrogen. concerns for global warming and pollution are exerting pressure Hydrogen is efficient, clean, and utilized for fuel cells as an energy to search for new renewable sources [1]. Biodiesel, methyl or ethyl source in portable electronics, power plants, and for the internal esters of fatty acids made from renewable biological resources, has combustion engine [6]. It has been reported that hydrogen can attracted much attention [1] because it generates less CO2 be produced by the pyrolysis of glycerol; however, this process compared to fossil fuels [2]. As a result, biodiesel production is requires high temperatures, and the product gas has a complex increasing; for example, the annual production capacity in the US composition [7]. Production of hydrogen by microorganisms may and the EU were 1.2 and 5.7 million metric tons in 2007 and will be more desirable than the current conventional thermal and elec- double by 2012 [3]. trolytic processes for hydrogen production since biological produc- Biodiesel is most often produced by reacting a fat or oil (triglyc- tion requires less energy [8]. erides) with methanol or ethanol in the presence of an alkali cata- Conversion of glycerol to hydrogen by photo-fermentation with lyst [2]. The production of 10 kg of biodiesel yields about 1 kg of Rhodopseudomonas palustris has been investigated [1]; however, glycerol [1]. The rapid growth of the biodiesel industry has created the long fermentation process (200–300 h) [1], the difficulty in excess glycerol, which has resulted in a significant decrease (about light penetration [9], and the difficulty in uniform light distribution 10-fold) in crude glycerol prices and has become a burden to the [9] hinder its practical use. Compared to photosynthetic processes, biodiesel industry [4]. fermentative hydrogen production generally gives two orders of Although crude glycerol can be used as boiler fuel and as a sup- magnitude higher rates of hydrogen production [8]. Moreover, plement for animal feed, the market value of crude glycerol is still anaerobic fermentation of glycerol to produce hydrogen is attrac- very low [4]. Thus, there is an urgent need to develop a practical tive because it produces not only hydrogen but also other biofuels process for converting glycerol to a useful product. Glycerol can such as ethanol [10]. Hydrogen production from glycerol by fer- be used as a carbon source by a number of microorganisms and mentation has been studied with Enterobacter aerogenes [10,11], Escherichia coli [12,13], Klebsiella pneumoniae [14], and mixed * Corresponding author. Fax: +1 979 865 6446. cultures [15]. However, hydrogen production is repressed at high E-mail address: [email protected] (T.K. Wood). glycerol concentrations and little ethanol is produced using 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.12.013 1034 H. Hu, T.K. Wood / Biochemical and Biophysical Research Communications 391 (2010) 1033–1038 E. aerogenes [10]. Although hydrogen production is improved by Materials and methods using an electrode system with thionine as an electron transfer mediator [11], the complex system may be impractical [1]. K. pneu- Bacterial strains, media, and growth conditions. The isogenic moniae has been investigated to produce 1,3-propanediol from E. coli BW25113 strains and plasmids used in this study are listed glycerol [4,5]; however, metabolic flux analysis shows that it in Table 1. For deleting and overexpressing genes, we used the Keio may not be suitable for hydrogen production because the produc- collection [24] and the ASKA library [25], respectively. Kanamycin tion of hydrogen and ethanol are dramatically decreased at higher (50 lg/mL) was used for pre-culturing the isogenic knock-outs. concentrations of glycerol [16]. In addition, industrial use of K. Chloramphenicol (30 lg/mL) was used for the strains harboring pneumonia would be limited because of its classification as an pCA24N and its derivatives. All experiments were conducted at opportunistic pathogen which can cause human infections [4]. Fur- 37 °C. thermore, mixed culture fermentations are very complex and are The glycerol medium used for hydrogen production was based on difficult to optimize due to the variable compositions of the micro- minimal medium that was supplemented with 10 g/L glycerol, 2 g/L bial communities [17]. tryptone, 5 mM sodium selenite, and 1.32 mM Na2HPO4 in place of Escherichia coli ferments glycerol anaerobically [12,13]; how- K2HPO4 [12]. The final concentration of MOPS (morpholinopropane- ever, it has a very low specific growth rate in glycerol [12], which sulfonic acid) was 50 mM, and the pH was adjusted to 6.3 by concen- is about 40-fold lower than aerobic growth in LB medium. None- trated NaOH. All chemicals were purchased from Fisher Scientific theless, E. coli is very promising for glycerol utilization because it (Pittsburgh, PA) and Sigma–Aldrich Co. (St. Louis, MO). is one of the most commonly used host organisms for metabolic Growth rates and the glycerol dehydrogenase assay. The specific engineering and industrial applications. It is easy to manipulate growth rates of the E. coli wild-type strain and the evolved mutants genetically, can produce a wide variety of anaerobic fermentation were determined in Luria–Bertani (LB) medium (aerobically) and products, and it is the best-characterized bacterium [6]. Further- glycerol medium (anaerobically) by measuring the turbidity at more, we have created E. coli bacteria capable of converting glucose 600 nm for two independent cultures of each strain as a function [6] and formate [18] into hydrogen using metabolic engineering as of time with values less than 0.7. The activity of glycerol dehydro- well as have used protein engineering for hydrogenase 3 [19] and genase for glycerol was measured as described previously [13] the hydrogen transcriptional activator FhlA [20] to improve hydro- using 100 mM potassium carbonate buffer (pH 9.4); this assay is gen yields. based on the change of absorbance at 340 nm due to the conver- Adaptive evolution is a set of environmentally-induced muta- sion of NAD+ into NADH by glycerol dehydrogenase. tions that confer growth advantages to the cell [21]. An organism Adaptive evolution and chemical mutagenesis. Adaptive evolution is subjected to serial or continuous cultivation for many genera- of BW25113 frdC was conducted anaerobically by successive batch tions to which it is not optimally adapted to select more fit genetic fermentations in glycerol medium supplemented with 50 lg/mL of variants [22]. Hence, the adaptive evolution method has been used kanamycin using sealed crimp-top vials (60 mL) with shaking at to select improved strains from E. coli [23]. 250 rpm for 78 passages. For each batch culture, the turbidity The aims of this study were to obtain a mutant that grows both was measured at 600 nm, cells were inoculated into vials with faster on glycerol and which has higher hydrogen productivity as fresh glycerol medium (20 mL), the vials were sealed, and the well as to investigate the mechanism by which these two traits headspace was purged with nitrogen for 5 min. The volume of are improved. By combining adaptive evolution with chemical the inoculum for each passage (5–50 lL) was adjusted due to mutagenesis, we obtained an evolved strain which has 5-fold faster changes in the growth rate of the evolved strain to ensure that cul- growth, 20-fold greater hydrogen productivity, and 5-fold greater tures would be in the exponential growth phase during the com- ethanol production. We also determined key enzymes that were plete fermentation. Cultures were frozen and stored at regular affected by the mutagenesis. intervals throughout adaptive evolution. Table 1 Escherichia coli bacterial strains, plasmids, and primers used in this study.