Bioresource Technology 108 (2012) 295–299 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Short Communication Effect of exogenous electron shuttles on growth and fermentative metabolism in Clostridium sp. BC1 ⇑ Venkata Nancharaiah Yarlagadda a,b, , Ashutosh Gupta a, Cleveland J. Dodge a, Arokiasamy J. Francis a,c a Environmental Sciences Department, Brookhaven National Laboratory, Upton, NY 11973, USA b Biofouling and Biofilm Processes Section, Water and Steam Chemistry Division, Bhabha Atomic Research Centre, Kalpakkam 603102, Tamil Nadu, India c Division of Advanced Nuclear Engineering, POSTECH, Pohang, South Korea article info abstract Article history: In this study, the influence exogenous electron shuttles on the growth and glucose fermentative metab- Received 12 October 2011 olism of Clostridium sp. BC1 was investigated. Bicarbonate addition to mineral salts (MS) medium accel- Received in revised form 8 December 2011 erated growth and glucose fermentation which shifted acidogenesis (acetic- and butyric-acids) towards Accepted 8 December 2011 solventogenesis (ethanol and butanol). Addition of ferrihydrite, anthraquinone disulfonate, and nicotin- Available online 16 December 2011 amide adenine dinucleotide in bicarbonate to growing culture showed no significant influence on fer- mentative metabolism. In contrast, methyl viologen (MV) enhanced ethanol- and butanol-production Keywords: by 28- and 12-fold, respectively with concomitant decrease in hydrogen, acetic- and butyric-acids com- Biobutanol pared to MS medium. The results show that MV addition affects hydrogenase activity with a significant Bioethanol Clostridium reduction in hydrogen production and a shift in the direction of electron flow towards enhanced produc- Electron shuttles tion of ethanol and butanol. Methyl viologen Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Anaerobic fermentative bacteria belonging to the genus Clos- tridia are of special interest in biofuel production from lignocellu- Electron shuttle (ES) compounds that cycle between oxidized losic biomass (Hess et al., 2011) due to their innate ability to and reduced states in multiple redox reactions play a vital role ferment 5 and 6 carbon sugars. Clostridium sp. BC1 (ATCC 53464) in bacterial metabolism. Exogenous supplements of ESs can cre- (henceforth referred to as BC1) isolated from an acidic metal con- ate alternate electron flows, thereby affecting the fermentation taminated site, has demonstrated efficient reduction of several re- pattern and biofuel production. Thus, exogenous ESs are added dox-active metals (i.e. U(VI), Pu(IV), Tc(VII), Fe(III) and Mn(IV)) in to accelerate electron transport and to modify the energy metab- mineral salts medium (Francis et al., 2008). BC1 was found to be olism and physiology of microorganisms. ESs such as neutral red, a rapid and efficient glucose fermenter compared to other Clostridia methylene blue, methyl viologen (MV) and benzyl viologen were strains, Clostridium acetobutylicum (ATCC 19403), Clostridium sphe- used to shift fermentative metabolism towards alcohol produc- noides (ATCC 19403), and Clostridium pasteurianum (ATCC 7040) tion (Datta and Zeikus, 1985; Peguin and Soucaille, 1995; Stein- (Gao and Francis, 2008). It was hypothesized that the electron busch et al., 2010; Tashiro et al., 2007). Addition of reduced shuttle compounds such as ferrihydrite (Fe(OH)3), nicotinamide anthraquinone disulfonate (AQDS) altered the fermentative pat- adenine dinucleotide (NADH), AQDS and MV cause a significant tern of Clostridium beijerinckii by increased H2 production (Hatch shift in the metabolic flow in the carbon metabolism of the metal and Finneran, 2008). Although the exact mechanism whereby ESs reducing bacterium BC1 from acidogenesis to soleventogenesis regulate the fermentative pathway is not fully understood, add- resulting in enhanced ethanol and butanol production. Fe(OH)3 is ing them to bacterial cultures is an attractive strategy for shifting a widespread ferric hydroxide and is an alternate electron acceptor the direction of electron flow from generating organic acids to under anaerobic conditions. AQDS is widely used as a humic acid alcohols (ethanol and butanol) and hydrogen (Reimann et al., analogue for investigating the role of quinone based ESs. NADH is 1996). the natural electron carrier present in the cells. MV has reduction potential closer to that of ferredoxin, which mediates electron transfer in a range of metabolic reactions. To understand the effect ⇑ of ES addition on the fermentative metabolism of BC1, we moni- Corresponding author at: Biofouling and Biofilm Processes Section, Water and tored the growth, glucose consumption and production of organic Steam Chemistry Division, Bhabha Atomic Research Centre, Kalpakkam 603102, Tamil Nadu, India. Tel.: +91 44 27480500x23342; fax: +91 44 27480097. acids (acetic-, butyric- and lactic-acids), alcohols (ethanol and E-mail addresses: [email protected], [email protected] (V.N. Yarlagadda). butanol) and gasses (H2 and CO2). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.040 296 V.N. Yarlagadda et al. / Bioresource Technology 108 (2012) 295–299 Fig. 1. Growth (OD 600 nm) and medium pH profiles during batch fermentation in mineral salts (MS) medium by Clostridium sp. BC1. Arrow indicates the addition of bicarbonate and electron shuttles prepared in bicarbonate at 24 h after inoculation. Error bars represent ±1SD. Error bars are not seen in some data points due to minimal variation between duplicates. 2. Methods sealed with aluminum caps and autoclaved. The culture was main- tained by inoculating the MS medium with 1-mL of log phase cul- 2.1. Electron shuttles ture and incubating at 26 °C. MV, NADH, and AQDS were obtained from Sigma–Aldrich. ES 2.3. Growth and fermentative metabolism solutions were prepared by dissolving 100 mg each of MV, NADH and AQDS in 40 mL of 1 M sodium bicarbonate. Fe(OH) was pre- 3 The serum bottles containing pre-reduced sterile MS medium pared as described previously (Dodge et al., 2002). ES solutions were inoculated with 1-mL of a log-phase culture (OD600 nm = 0.4) were purged with nitrogen gas for 10 min and stored under nitro- and incubated at 26 °C. A 1.5-mL of HCOÀ or ES in HCOÀ was added gen atmosphere in serum bottles. HCOÀ solution was also stored 3 3 3 to the culture after 24 h of growth. The final concentration of MV, under similar conditions. NADH, AQDS and Fe(OH)3 in the culture was 0.36, 0.13, 0.23 and 0.56 mM, respectively. All the treatments were performed in trip- licates. Periodically, 5 mL of the culture was withdrawn and ana- 2.2. Culture and growth conditions lysed for optical density, pH, organic acids and alcohols. BC1 was grown in the mineral salts (MS) medium containing 1% (w/v) glucose as described previously (Nancharaiah and Francis, 2.4. Chemical analysis 2011). The medium was pre-reduced by boiling for 10 min while purging with ultra-high-purity nitrogen gas dispensed as 40-mL At periodic intervals, total gas production was determined by aliquots into 60-mL serum bottles in an anaerobic chamber (Coy measuring the head space for pressure using a pressure gauge con- laboratory products, USA), fitted with butyl rubber stoppers, crimp nected to syringe needle. The CO2 and H2 concentrations in the V.N. Yarlagadda et al. / Bioresource Technology 108 (2012) 295–299 297 headspace were measured by gas chromatography using a Gow mined using a Beckman U350 pH meter with a Beckman 511275- Mac series 580 gas chromatograph under isothermal mode using AB combination electrode. The culture sample was then filtered thermal conductivity detector. The culture medium pH was deter- through a 0.45 lm Millex filter, and glucose, alcohols and organic Table 1 Comparison of the production of various fermentation products by Clostridium sp. BC1 grown in mineral salts (MS) medium with and without bicarbonate and ESs. The metabolite concentrations were determined after 96 h of incubation. Substrate or product (mM) Medium MS NaHCO3 NaHCO3 Fe(OH)3 AQDS NADH MV Glucose utilized 32.3 54.9 55.0 55.0 55.0 51.5 Total organic acids 41.7 75.7 75.3 77.9 74.6 36.7 Acetic acid 25.5 62.8 63.1 64.6 62.4 29.2 Butyric acid 15.5 12.2 11.5 12.6 11.4 1.0 Lactic acid 0.7 0.7 0.7 0.7 0.8 6.5 Total solvents 2.8 17.4 18.5 17.4 19.7 48.4 Butanol 2.0 12.9 13.5 12.8 14.1 24.9 Ethanol 0.8 4.5 5.0 4.6 5.6 23.5 Fig. 2. Production of metabolites (acids and alcohols) during batch fermentation by Clostridium sp. BC1 in mineral salts (MS) medium. Arrow indicates the addition of bicarbonate and ES in bicarbonate at 24 h after inoculation Error bars represent ±1SD. Error bars are not seen in some data points due to minimal variation between duplicates. 298 V.N. Yarlagadda et al. / Bioresource Technology 108 (2012) 295–299 acids were analyzed by high pressure liquid chromatography using The increase in ethanol and butanol levels were 5- and 2-fold, À a Shimadzu LC-10AS liquid chromatography fitted with a Bio-Rad respectively compared to HCO3 . This difference in product distri- HPX-87H column. A diluted sulphuric acid (0.003 N) was used as bution with different ESs could be attributed to the differences in mobile phase at a flow rate of 0.7 mL minÀ1. Glucose, alcohols the reduction potentials of ESs used in this study. Since MV has and organic acids were determined using a refractive index and a the closest reduction potential to that of ferredoxin, the natural UV–vis detector, respectively. electron carrier in Clostridia, only MV could possibly compete with ferredoxin and cause changes in carbon and electron flow. The yields of butanol (0.48) and ethanol (0.46) with MV addition were 3.