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Journal of Bioscience and BioengineeringVOL. 115 No. 2, 173e175, 2013

NOTE

Decreased hydrogen production leads to selective butanol productionin co-cultures of Clostridium thermocellum and Clostridium

saccharoperbutylacetonicum strain N1-4

Shunichi Nakayama,* Yukiko Bando, Akihiro Ohnishi, Toshimori Kadokura, and Atsumi Nakazato

Department of Fermentation Science and Technology, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received 3 April 2012; accepted 26 August 2012Available online 19 September 2012

* CorrespondE-mail add

1389-1723/$http://dx.doi

When Clostridium thermocellum and Clostridium saccharoperbutylacetonicum strain N1-4 were co-cultured hydrogenproduction decreased and butanol was selectively produced with extremely low level of acetone. Since the high butanolproduction correlates with low hydrogen production, the molecular selection of hydrogenase gene activity is expectedto yield strains exhibiting a higher butanol ratio.

� 2012, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Butanol production; Hydrogen production; Clostridium species; Hydrogenase; Co-culture]

Biobutanol is an attractive biofuel because it has an energydensity similar to that of gasoline and is expected to be a sustain-able resource for biofuel production (1,2). Since butanol-producingClostridium spp. simultaneously produces acetone that is chemi-cally similar to butanol (3), separation of these compounds isdifficult and cost-ineffective (4). Although the disruption ofacetone-producing genes leads to reduced acetone production andincreased butanol production, other by-products such as acetate orethanol are produced instead of acetone (5,6). This is thought to bedue to the lack of reducing power because the butanol-producingpathway requires NADH as a reducing agent but sufficient NADH isnot available during fermentation.

We previously described butanol production from Avicel cellu-lose by the co-culturing of butanol-producing Clostridium saccha-roperbutylacetonicum strain N1-4 and cellulose-degradingClostridium thermocellum NBRC103400 (7). This co-culture systemmainly produces butanol and the acetone level is extremely lowthough the enzymatic activity related to acetone production isunaltered.

It has also been reported that the modification of electron flowimproves the ratio of acetone and butanol produced. Butanolproduction is enhanced by an increase in hydrogen partial pressureor by adding an artificial electron carrier (8e10). On the other hand,downregulation of H2-uptake hydrogenase hupCBA gene expres-sion results in increased acetone and hydrogen production anddecreased butanol production (11). Thus, we assumed that the lowacetone production observed in the co-culture system would bedue to the modification of electron flow because the culture

ing author. Tel./fax: þ81 3 5477 2382.ress: s3nakaya@nodai.ac.jp (S. Nakayama).

e see front matter � 2012, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2012.08.020

conditions would become more reduced by the dissolved hydrogengas produced by C. thermocellum during cellulose metabolism.

In this report we compare the hydrogen metabolism of strainN1-4 in single- and co-culture states to elucidate the high butanolratio. We found a correlation between low hydrogen and butanolproduction due to a lower oxidation-reduction potential (ORP).

The butanol-producing C. saccharoperbutylacetonicum strainsN1-4 (ATCC 13564) and N1-4hup� (NRIC 0816) harboring thehupC antisense RNA expressing plasmid (11) were anaerobicallygrown in TYAmedium (6 g Tryptone Peptone, 2 g yeast extract, 40 gglucose, 3 g CH3COONH4, 0.3 g MgSO4$7H2O, 0.5 g KH2PO4, 0.01 gFeSO4$7H2O per liter, pH 6.5) at 30�C. For fermentation analysis insingle-culture, the strains were cultured in a 250-ml glass bottlewith 100 ml TYA medium equipped with a rubber stopper. Theinitial cell densities were adjusted to OD600 nm ¼ 0.1 after collectionof the cells grown for 24 h, washed and suspended in TYA medium.Twenty micrograms per milliliter of chloramphenicol was added tothe culture medium when the strain N1-4hup� was cultivated.

C. thermocellum NBRC103400 (ATCC 27405) was culturedanaerobically at 60�C as previously described (7). Co-cultureexperiments were performed in a 250-ml glass bottle equippedwith a rubber stopper. C. thermocellum cells grown on NBRCmedium 979 [1.3 g (NH4)2SO4, 2.6 g MgCl2$6H2O, 1.43 g KH2PO4,7.2 g K2HPO4$3H2O, 0.13 g CaCl2$2H2O, 6 g sodium glycer-ophosphate, 1.1 mg FeSO4$7H2O, 0.25 g glutathione, 4.5 g yeastextract, 1 mg Resazurin per liter, pH 7.0] containing 5 g/L of cello-biose at 60�C were collected by centrifugation. Cell pellets werewashed and suspended in the same medium without any carbonsource added and adjusted to OD600 nm ¼ 0.2. Fifty ml of the cellsuspension was then used to inoculate 50 ml of NBRC medium 979containing 80 g/L of Avicel cellulose (final OD600 nm ¼ 0.1 and finalconcentration of Avicel cellulose is 40 g/L) and cultured at 60�C for

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TABLE 1. Enzymatic activities of hydrogenases of strain N1-4.

Enzymes Single-culture Co-culturea Hydrogenaseactivity ratiob

Hydrogen-evolving activity 44.14 � 2.92 62.40 � 3.43 0.71Hydrogen-uptake activity 0.05 � 0.00 0.07 � 0.00 0.68

a Activities were normalized based on thiolase activity which is specific to strainN1-4 because the crude extracts prepared from co-cultures contained enzymesproduced by both strain N1-4 and C. thermocellum as previously described (7). Theactual thiolase activities (mU/mg) prepared from single- and co-cultures were53.3 � 2.6 and 95.0 � 11.0, respectively.

b The ratio is evaluated by dividing the value of single-culture by that of co-culture.

174 NAKAYAMA ET AL. J. BIOSCI. BIOENG.,

24 h. The butanol-producing Clostridium strains grown for 24 hwere collected by centrifugation, washed, and suspended in TYAmedium and adjusted to OD600 nm ¼ 1.1. Ten ml of cell suspensionwas then added to the bottle containing C. thermocellum culturesafter the cultivation temperature was decreased to 30�C (finalOD600 nm ¼ 0.1). The subsequent co-cultures were incubated at30�C.

The gaseous products were collected in gas sampling bag AAK-1(GL Science Inc., Japan) through the glass tube embedded in therubber stopper. The gaseous fermentation products and celluloseconcentrations were quantified as previously described (7).

Butanol [9.0 g/L (120.7 mM)] and 3.0 g/L (52.5 mM) of acetonewere produced by strain N1-4 consuming 40 g/L of glucose for 5days, while the co-culture system produced 5.7 g/L (76.8 mM) ofbutanol and extremely low levels of acetone (4.6 mM) consuming31.7 g/L of crystalline cellulose for 12 days cultivation (Fig. 1a). Theacetone and butanol yields (mol/mol) evaluated by concentration(mol) of produced acetone and/or butanol per consumed glucose(mol) in the single-culture were 0.24 and 0.54, respectively. On theother hand, the acetone and butanol yields (mol/mol) evaluated byconcentration (mol) of produced acetone and/or butanol perglucose equivalent of cellulose consumed (mol) in the co-culturewere 0.03 and 0.43, respectively, indicating that hardly any acetonewas produced in the co-culture system. The hydrogen gas producedby the co-culture of strain N1-4 and C. thermocellum (3.0 mmol)was 71.4% lower than that produced from the glucose-grown strainN1-4 (10.5 mmol). The hydrogen yield (mol/mol) of the co-culturesystem (0.17) was 64% lower than that of strain N1-4 alone (0.47). Inaccordance with a previous report (9), the low acetone productioncorrelated well with the decreased hydrogen gas production.

Since decreased hydrogen gas production is thought to reflectdecreased hydrogenase activity (9), to elucidate the low hydrogenproduction in the co-culture system we compared the hydrogen-evolving and hydrogen-uptake activity of strain N1-4 in single- andco-culture states according to the method of Vasconcelos et al. (12)(Table 1). Since strain N1-4 has both activities (11), it was expectedthat lower hydrogen-evolving activity or higher hydrogen-uptakeactivity would occur in the co-culture system. Both hydrogen-evolving and hydrogen-uptake activities in co-culture were slightlyhigher than those in single-culture. However, the ratio of hydrogen-evolving and hydrogen-uptake activities was not significantlydifferent. This result indicates that hydrogenase activity is compa-rable in both single- and co-culture, and that the low hydrogenproduction is not dependent on the hydrogenase activity of strainN1-4.

FIG. 1. Fermentation products (a) and hydrogen production (b) from 40 g/L of glucoseby strain N1-4 cultivated for 5 days, and 40 g/L of Avicel cellulose by strain N1-4, co-cultured with C. thermocellum cultivated for 12 days. Bars show amounts of acetate(horizontal stripes), butyrate (vertical stripes), ethanol (open), acetone (gray) andbutanol (closed). Values as shown as the mean of samples assayed in triplicate; bars,standard error.

The other possibility examined is that the decreased ORP due tothe dissolved hydrogen gas produced by C. thermocellum physico-chemically inhibits the hydrogen gas production and the electronflow leading to NADH generation. As reported by Wang et al.,decreased ORP leads to high butanol production and low acetoneproduction (13). Thus, wemeasured the ORP by TPX-999i equippedwith an ORP electrode (Tokyo Chemical laboratories Co., Ltd, Japan)calibrated with quinhydrone solution as a redox standard. The ORPof co-cultures and single-cultures cultivated for 24 h were�510.5� 6.5 mV and�473.5� 6.5 mV, respectively, and the ORP ofthe co-culture system was sustained at 35 mV lower than that ofthe single-culture throughout the cultivation (P < 0.01). Theseresults clearly indicate that it was not the hydrogenase activity ofbutanol-producing strain N1-4, but the reducing conditions createdby C. thermocellum that was attributable for the low hydrogen andhigh butanol production because electron flow leads to NADHgeneration and the increased reducing power is used in thebutanol-producing pathway.

To further confirm the effect of co-culture with C. thermocellumon butanol production, the hupCBA mutant strain (strain N1-4hup�), in which expression of the hydrogen-uptake hydrogenasegene (hupCBA) is downregulated by antisense RNA against hupCand therefore exhibits higher hydrogen production and lowerbutanol production compared to parental strain N1-4 (11), wasexamined. 7.5 g/L (101.1 mM) of butanol and 5.2 g/L (88.7 mM) ofacetone were produced by strain N1-4hup� consuming 40 g/L ofglucose for 5 days, while the co-culture system produced 4.5 g/L(60.9 mM) of butanol and 1.5 g/L (25.2 mM) acetone afterconsuming 32.4 g/L of crystalline cellulose for 12 days cultivation(Fig. 2). The acetone and butanol yields (mol/mol) in single-culturewere 0.40 and 0.46, respectively. On the other hand, the acetoneand butanol yields (mol/mol) in co-culture were 0.14 and 0.35,

FIG. 2. Fermentation products (a) and hydrogen production (b) from 40 g/L of glucoseby strain N1-4hup� cultivated for 5 days, and 40 g/L of Avicel cellulose by strain N1-4hup�, co-cultured with C. thermocellum cultivated for 12 days. Bars show amounts ofacetate (horizontal stripes), butyrate (vertical stripes), ethanol (open), acetone (gray)and butanol (closed). Values as shown as the mean of samples assayed in triplicate;bars, standard error.

VOL. 115, 2013 NOTE 175

respectively, indicating that acetone productionwas also decreasedin co-culture system. Hydrogen production in co-culture with N1-4hup� and C. thermocellum was 35.3% lower than that producedfrom glucose by strain N1-4hup�. The hydrogen yield (mol/mol) inthe co-culture system (0.43) was 57% lower than that from strainN1-4hup� (1.00). Interestingly, the co-culture system using strainN1-4hup� also exhibited lower hydrogen and acetone productionand high butanol production. The ORP of co-cultures and single-cultures cultivated for 24 h were �527.0 � 1.2 mV and�469.8 � 5.8 mV, respectively, and the ORP of the co-culturesystemwas sustained at 57mV lower than that of the single-culturethroughout the cultivation (P < 0.01). Thus, we conclude that co-culture with the hydrogen-producing C. thermocellum leads to lowhydrogen production and high butanol production in the butanol-producing strain N1-4.

In summary, we describe low hydrogen production that is attrib-utable to the high butanol production in co-cultures of strain N1-4.Since the disruption of acetone-producing genes leads to other by-products due to a lack of reducing power, themodification of electronflowwould be a promising target for selective butanol production instrain N1-4. Thus, the engineering of low hydrogen-producing,butanol-producing Clostridia bymolecular selection for hydrogenaseactivity is expected to yield strains exhibiting a higher butanol ratio.

This study was supported in part by a Grant-in-Aid for Young Scientists (B).

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