Effect of the Reducing Agent Dithiothreitol on Ethanol and Acetic Acid ...

Effect of the Reducing Agent Dithiothreitol on Ethanol and Acetic Acid Production by Clostridium Strain P11 Using Simulated Biomass-Based Syngas B. Kubandra Babu, H. K. Atiyeh, M. R. Wilkins, R. L. Huhnke* ABSTRACT. The effect of dithiothreitol (DTT) on enhancing ethanol production from syngas using Clostridium strain P11 (ATCC PTA-7826) was investigated in 250 mL serum bottles. Reducing agents help in regeneration of NADH from NAD+. NADH is utilized in the production of alcohol from aldehydes. The effect of DTT was studied in two different media: 0.1% (w/v) yeast extract, and 1% (w/v) corn steep liquor. Strain P11 was fed with syngas every 24 h, and samples were collected to measure pH, cell mass, and product concentrations. Various concentrations of DTT were examined. Results showed more than a 350% increase in ethanol concentration in media that contained at least 7.5 g L-1 of DTT after 360 h of fermentation compared to the control medium (without DTT) in 0.1% (w/v) yeast extract medium. However, only a 35% increase in ethanol production was noticed in 1% (w/v) corn steep liquor in the presence of 2.5 and 5.0 g L-1 of DTT compared to the control medium. The results suggested that the use of small concentrations of DTT in the broth enhances ethanol production from syngas. Improvement in ethanol production efficiency will increase the cost-effectiveness of the syngas fermentation technology. Keywords. Clostridium, Dithiothreitol, Ethanol, Fermentation, Reducing agent, Syngas.

L

iquid fuel is the lifeline of the transportation industry. The decrease in petroleum imports and increasing greenhouse gas (GHG) emissions has made innovation in the field of biofuels a national priority (Dale, 2003). The use of ethanol as a fuel in the transportation industry increased by nearly 700% in the last ten years (RFA, 2009), and the production of ethanol increased by about 36% each year between 2006 and 2008 (RFA, 2009). Ethanol production in the U.S. increased from about 9.2 billion gallons in 2008 to 10.6 billion gallons by the end of 2009 (RFA, 2010). This quite clearly shows that the production of fuel-grade ethanol in the U.S. is increasing with every passing year. The Energy Independence and Security Act of Submitted for review in April 2010 as manuscript number BE 8528; approved for publication by the Biological Engineering Editorial Board of ASABE in October 2010. The authors are Balaji Kubandra Babu, Graduate Research Assistant, Hasan K. Atiyeh, ASABE Member Engineer, Assistant Professor, Mark R. Wilkins, ASABE Member Engineer, Assistant Professor, and Raymond L. Huhnke, ASABE Fellow, Professor, Department of Biosystems and Agricultural Engineering, Oklahoma State University, Stillwater, Oklahoma. Corresponding author: Hasan K. Atiyeh, Department of Biosystems and Agricultural Engineering, Oklahoma State University, Stillwater, Oklahoma 74078; phone: 405-744-8397; fax: 405-744-6059; e-mail: [email protected]. Biological Engineering 3(1): 19-35

© 2010 ASABE ISSN 1934-2799

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2007 has mandated that, by the year 2022, the U.S. must produce 36 billion gallons of biofuels per year (EPA, 2009), making innovation in biofuels production a top priority. The gasification-fermentation platform is one promising technology for conversion of cellulosic feedstocks to ethanol. In this platform, biomass is pyrolyzed to produce synthesis gas (syngas) primarily containing CO, CO2, H2, and N2, which is fed to bacteria to produce ethanol. The main advantage of employing syngas fermentation for ethanol production is that a wide spectrum of raw materials can be gasified to produce syngas. By utilizing lignocellulosic feedstocks for fuel production, corn and other food crops are spared. Syngas components can be fermented by anaerobes such as Clostridium ljungdahlii (Klasson et al., 1992; Phillips et al., 1994; Younesi et al., 2005), Clostridium autoethanogenum (Abrini et al., 1994), Clostridium carboxidivorans P7 (Ahmed et al., 2006), and Clostridium strain P11 (Tanner, 2008; Huhnke et al., 2010) to produce acetic acid and ethanol. C. carboxidivorans P7 (Ahmed et al., 2006) and Clostridium strain P11 (Tanner, 2008; Huhnke et al., 2010) can also produce butanol from syngas. Henstra et al. (2007) reviewed other microorganisms that can grow on syngas for biofuel production. Clostridium bacteria produce ethanol and acetic acid through the Wood-Ljungdahl pathway, also called the acetyl-CoA pathway (Wood et al., 1986). In the Wood-Ljungdahl pathway, H2 serves as an electron donor, while CO2 serves as the electron acceptor. There are two main steps involved in the production of end products like ethanol, acetic acid, and butanol from CO. In the first step, two individual pathways separately produce the carbonyl and the methyl precursors of acetyl-CoA (each pathway produces one type of precursor). In the second step, the two precursors are condensed to form acetyl-CoA, which is then used for the production of either acetic acid or ethanol based on the conditions prevailing within the microorganism. The pathway involved in the conversion of acetyl-CoA to acetic acid is called acidogenesis, and the pathway for conversion of acetyl-CoA to ethanol and other non-polar solvents is called solventogenesis. The reactions involved in the production of acetic acid from acetyl-CoA are shown below: Acetyl-CoA + Pi → Acetyl-phosphate + Pi

(1)

Acetyl-phosphate + ADP → Acetic acid + ATP

(2)

Ethanol is produced from acetyl-CoA according to the following reactions: Acetyl-CoA + NADH + H+ → Acetaldehyde + NAD+ + CoA-SH

(3)

Acetaldehyde + NADH + H+ → Ethanol + NAD+

(4)

Factors such as pH, availability of nutrients, and reducing equivalents can influence the metabolic shift from acidogenesis to the solventogenesis phase during syngas fermentation (Ahmed et al., 2006; Vasconcelos et al., 1994). In addition, the gas-liquid mass transfer rates and media components are very important factors that affect cell growth rate and product formation during syngas fermentation. The addition of reducing agents to fermentation media was reported to enhance solventogenesis (Peguin et al., 1994; Rao and Mutharasan, 1986). Reducing agents act as artificial electron carriers that donate electrons in a redox reaction and as a result are oxidized. The donated

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Biological Engineering

electrons can be used to reduce NAD+ to NADH or NADP+ to NAD(P)H. NADH is used by acetogens to produce ethanol from acetyl-CoA, as shown in equations 3 and 4 (Ahmed, 2006). Methyl viologen has been observed to increase ethanol production in Clostridium acetobutylicum by diverting the electron flow towards regeneration of NADH from NAD+ (Rao and Mutharasan, 1986). The addition of 1 mM methyl viologen to fermentation medium increased production of butanol by 0.2 M per 1 M glucose consumed by C. acetobutylicum (Peguin et al., 1994). This was due to the increase in NAD(P)H formation, which was utilized in production of butanol. Addition of 0.1 mM methyl viologen enhanced ethanol production by 160% during syngas fermentation using Clostridium strain P11, while the addition of 0.1 mM neutral red increased ethanol production by 22% as compared to treatments with no reducing agent (Panneerselvam, 2009). The maximum ethanol concentrations observed with addition of methyl viologen and neutral red were 1.3 and 0.6 g L-1, respectively. Benzyl viologen at 0.1 mM was shown to inhibit solventogenesis (Panneerselvam, 2009). Dithiothreitol (DTT), also called Cleland’s reagent, is a water-soluble reducing agent with an oxidation-reduction potential (ORP) of -332 mV at pH 7 and -366 mV at pH 8.1 (Cleland, 1964). It is expected that DTT can favor alcohol production in syngas fermentation in more than one way. DTT is a reducing agent and hence can donate electrons (Krasnovsky et al., 1980). Electrons are required for the regeneration of NADH from NAD+, which in turn favors alcohol production (Rao and Mutharasan, 1989). DTT also can protect thiol groups (SH) in some of the key enzymes involved in alcohol fermentation from oxidation during yeast fermentation (Asada et al., 1981). This might enhance alcohol production from syngas as well if similar enzymes in syngas fermenting bacteria are also protected. More negative ORP favors alcohol production (Frankman, 2009). Addition of DTT significantly reduced the ORP of fermentation broth used for 4-decanolide production by Sporidiobolus (Wang et al., 2000). Hence, it is hypothesized that DTT can favor alcohol production by reducing the ORP of fermentation broth. The present work reports the effect of DTT on ethanol and acetic acid production from syngas using Clostridium strain P11.

Materials and Methods Microbial Catalyst and Culture Medium Clostridium strain P11 (ATCC PTA-7826) provided by Dr. Ralph Tanner, University of Oklahoma, was used as the microbial catalyst. The microbial culture was grown under strict anoxic conditions in two different fermentation media: yeast extract (YE) and corn steep liquor (CSL). The same optimized media components used by Saxena (2008) for maximum ethanol production with Clostridium strain P11 were used in the current study. CSL is rich in carbohydrates, vitamins, minerals, and trace metals. It is also a lower-cost nutrient compared to YE, the primary media constituent currently being used in syngas fermentation. The media consisted (per liter) either 1 g yeast extract (Difco Laboratories, Detroit, Mich.) or 10 g corn steep liquor (Sigma Aldrich, St Louis, Mo.) depending on the type of fermentation medium used and the following components: 10 g morpholinoethanesulfonic acid (MES), 1 mL resazurin (0.1%), 30 mL minerals stock solution, 10 mL vitamins stock solution, 10 mL trace metals stock solution, and 10 mL of 4% cysteine sulfide solution. MES and resazurin were 3(1): 19-35

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used as biological buffer and redox indicator, respectively. The initial pH of the media was adjusted to 6.0 using 2N KOH before inoculation. The compositions of the fermentation media were similar to the growth media. The minerals stock solution consisted of the following components (per liter): 100 g ammonium chloride, 4 g calcium chloride, 20 g magnesium sulfate, 10 g potassium chloride, and 10 g potassium phosphate monobasic. The trace metal stock solution consisted of the following components (per liter): 2 g nitrilotriacetic acid, 1 g manganese sulphate, 0.8 g ferrous ammonium sulphate, 0.2 g cobalt chloride, 1 g zinc sulphate, 0.2 g nickel chloride, 0.02 g sodium molybdate, 0.1 g sodium selenate, and 0.2 g sodium tungstate. The vitamins stock solution consisted of the following components (per liter): 0.005 g p-(4)-aminobenzoic acid, 0.002 g d-biotin, 0.005 g pantothenic acid (calcium salt), 0.002 g folic acid, 0.01 g MESNA, 0.005 g nicotinic acid, 0.01 g pyridoxine, 0.005 g riboflavin, 0.005 g thiamine, 0.005 g thioctic acid, and 0.005 g vitamin B-12. Simulated Syngas Commercial syngas composed of 5% H2, 15% CO2, 20% CO, and 60% N2 by volume (Airgas, Tulsa, Okla.) was used in this study. This gas composition was similar to the composition of syngas obtained by gasifying switchgrass in a fluidized bed gasifier (Rajagopalan et al., 2002). Batch Studies Batch experiments were done in 250 mL serum bottles (Wheaton, N.J.) with 100 mL of fermentation medium, which was prepared and sparged with nitrogen. The media were then dispensed into serum bottles inside a glove box under strict anoxic (anaerobic) conditions. This was followed by the addition of 1 mL of 4% cysteine sulfide solution per 100 mL media and sterilization of the media in an autoclave (Primus Sterilizer Co. Inc., Omaha, Neb.). After the bottles were cooled to room temperature, DTT was added using a syringe fitted with a sterile 0.2 micron nylon filter. The serum bottles were then fed with syngas at 239 kPa (absolute) (i.e., 16.6 mmole of syngas) and inoculated with 5% (v/v) of Clostridium strain P11 culture. Four concentrations of DTT (2.5, 5.0, 7.5 and 10.0 g L-1) and a control (with no DTT) were used in this study. Each treatment was repeated four times. The bottles were placed on a rotary shaker (Innova 2100, New Brunswick Scientific, Edison, N.J.) at 150 rpm and incubated at 37ºC in a temperature-controlled room. The fermentation was monitored for 360 h. Liquid samples of 2.0 mL were withdrawn every 24 h from the bottles under aseptic conditions in a biosafety cabinet using a sterile syringe. Headspace gas was purged from the bottle and replaced with fresh syngas at 239 kPa (absolute) each day after the liquid samples were collected from the serum bottle. Sterile 0.2 μm PTFE (polytetrafluoroethylene) membrane filter (VWR International, West Chester, Pa.) was used in the syngas inlet line. The total syngas that was added in the headspace in each fermentation medium after 360 h was 269 mmole. Cell concentration, pH, acetic acid, ethanol, and butanol concentrations were measured for each sample. Analytical Procedures The optical density (OD) of samples was measured at 660 nm using a UV-Vis spectrophotometer (Cary 50 Bio, Varian, Inc., Palo Alto, Cal.). Samples with OD val-

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ues above 0.4 were diluted so that the OD value was within the linear range of the calibration curve between cell mass and OD (cell mass, g L-1 = 0.396 × OD - 0.0521). The calibration curve was established using the dry weight method as described by Panneerselvam (2009). Acetic acid and butanol in the samples were analyzed using a gas chromatograph (GC 6890, Agilent Technologies, Wilmington, Del.) fitted with a PoraPak QS 80/100 column. The GC was operated at 210ºC with helium as the carrier gas. Chromatograms were analyzed using ChemStation data analysis software (Agilent Technologies, Wilmington, Del.). Ethanol concentration was measured using a YSI 2700 Biochemistry analyzer (YSI Life Sciences, Yellow Springs, Ohio) using immobilized alcohol oxidase in the enzyme membrane. Statistical Anaylsis An analysis of variance was calculated using SAS (release 9.2, SAS Institute, Inc., Cary, N.C.). To test if DTT concentration has a significant effect on the fermentation process in each medium, ethanol concentration was the dependent variable and DTT concentration was the independent variable. Dunnett’s test (Dunnett, 1955) at 95% confidence level was used to compare pH, cell mass, ethanol, and acetic acid concentrations from each DTT concentration to those from the control at each time point. To test if both DTT concentration and media type have a significant effect on the fermentation process, ethanol and acetic acid concentrations after 360 h were the dependent variables and DTT concentration and media type were the independent variables. A protected least significant difference test (Dunnett, 1955) at 95% confidence level was used to compare the concentrations of ethanol and acetic acid at 360 h from each DTT concentration and media type.

Results and Discussion Cell Growth Growth profiles of Clostridium strain P11 in YE media containing various concentrations of DTT were similar until 168 h (fig. 1a). Cells were in the exponential phase in the first 144 h, after which cells entered the deceleration phase, followed by the stationary phase. However, cell concentration declined after 192 h in YE media without DTT. A maximum cell mass concentration of 0.37 g L-1 was observed after 192 h in YE media with a DTT concentration of 2.5 g L-1. The difference in cell mass concentration in the first 168 h for all DTT concentrations tested was statistically insignificant (p > 0.05). However, a clear difference in cell mass concentration was noticed between YE media without DTT and with DTT after 192 h. The specific growth rate for Clostridium strain P11 decreased with the increase in DTT concentration (table 1). Cells grown in CSL media were in the exponential growth phase for the first 48 h (fig. 1b). Cells in media with 0, 2.5, and 5 g L-1 DTT remained in the stationary phase from 96 h to 288 h. However, cells in treatments with 7.5 and 10 g L-1 DTT remained in the stationary phase from 48 h to 120 h, after which cell death was observed. It is clear that addition of higher concentrations of DTT (7.5 g L-1 or higher) reduced the final cell concentration. The maximum cell concentration obtained was 0.45 g L-1 in the control treatment after 264 h. After 120 h of fermentation, treatments with 7.5 and 10 g L-1 DTT produced less cell mass compared to the control (p < 0.05).

3(1): 19-35

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Cell Mass (g L-1 )

(a)

= = = = =

0 g L -1 2.5 g L -1 5 g L -1 7.5 g L -1 10 g L -1

Cell Mass (g L -1 )

(b)

= 0 g L -1 = 2.5 g L -1 = 5 g L -1 = 7.5 g L -1 = 10 g L-1

Figure 1. Kinetics of cell mass production in (a) 0.1% YE media and (b) 1% CSL media containing various concentrations of dithiothreitol (0, 2.5, 5, and 7.5 and 10 g L-1). Error bars show typical experimental uncertainty.

It should be mentioned that Clostridium strain P11 grew faster in CSL media compared to YE media, as indicated by the higher specific growth rates in CSL media with all DTT treatments (table 1). This could be due to the presence of more nutrients in the CSL media. In addition, DTT concentrations above 3 g L-1 were shown to inhibit the growth of Escherichia coli because DTT affected the intracellular protein folding and disulfide bond formation (Missiaka et al., 1993; Gill et al., 1998). However, in the present study, Clostridium strain P11 grew in both YE and CSL media containing over three times more DTT than with E. coli. 24


[a]

Table 1. Specific growth rates and ethanol yields in 0.1% YE and 1% CSL media with various dithiothreitol concentrations using Clostridium strain P11. Specific Growth Initial DTT Initial Cells Ethanol Yield[a] -1 -1 -1 Medium (g L ) (mg L ) Rate, μ (h ) (g g-1) 0.1% YE 0.0 19.3 0.035 3.48 2.5 18.9 0.033 2.98 5.0 21.5 0.028 3.60 7.5 20.1 0.022 7.60 10.0 22.4 0.022 11.42 1% CSL 0.0 38.5 0.042 5.31 2.5 38.5 0.078 8.33 5.0 37.0 0.074 9.16 7.5 30.4 0.077 15.08 10.0 29.5 0.043 16.24 Values (g ethanol g-1 cell mass) are based on average ethanol and cell mass concentrations after 360 h.

pH and Pressure Profiles The pH values of the YE media with all tested DTT concentrations decreased similarly with time (fig. 2a). This was largely influenced by the production of acetic acid. The pH of the media decreased from 6 to about 4.5 in the first 264 h. It then increased to about 4.8 between 264 and 360 h, which was also the time at which ethanol production rate increased. This indicates that ethanol production occurs during the stationary phase in the pH range of 4.5 to 4.8. The difference in pH profiles in YE media with all tested DTT concentrations was statistically insignificant (p > 0.05). The pH profiles during the course of fermentation in CSL media were similar for all DTT treatments from 0 h to 48 h, as shown in figure 2b. The pH dropped from an initial value of 6 to about 5.4 in 48 h. However, the pH change was different for different treatments from 48 h to 360 h. In the control, the pH continued to decrease from 5.4 (at 48 h) to about 5.0 (at 120 h). This was due to the increase in acetic acid concentration in the fermentation broth with time. After this, the pH increased to about 6.0 (at 360 h). This was quite clearly due to consumption of acetic acid, which could have been used for ethanol production. In the CSL medium with 10 g L-1 DTT, the pH reached a value of 6 after 144 h of fermentation, as almost all the acetic acid in the broth had been consumed by the bacteria. The pH profile for the control and 2.5 and 5 g L-1 DTT treatments were similar after 216 h. Figure 3a shows changes in headspace pressure in the fermentation bottles with YE media after every 24 h. Initial pressure was set to 239 kPa (absolute) (i.e., 16.6 mmole of syngas) by purging the headspace every 24 h after taking samples from all media to determine product and cell mass concentrations. More syngas was consumed in the YE media in the first 192 h (most of the growth phase) compared to the rest of the fermentation period (stationary and death phases). The increase in syngas consumption from 24 to 96 h was due to the increase in cell mass concentration, which required more substrate for growth and product formation. Almost no gas consumption was noticed after 264 h in the YE media with DTT concentrations below 5 g L-1. However, syngas consumption was noticed in the YE media with DTT above 7.5 g L-1. This indicates higher cell activity in YE media with 7.5 g L-1 DTT, which favored ethanol production, as will be explained in the next section.

3(1): 19-35

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(a) = 0 g L -1 = 2.5 g L -1 = 5 g L -1 = 7.5 g L -1 = 10 g L-1

(b)

= 0 g L -1 = 2.5 g L -1 = 5 g L -1 = 7.5 g L -1 = 10 g L-1

Figure 2. pH profiles during syngas fermentation in (a) 0.1% YE media and (b) 1% CSL media containing various concentrations of dithiothreitol (0, 2.5, 5, 7.5, and 10 g L-1). Error bars show typical experimental uncertainty.

The change in headspace pressure in the serum bottles during the course of fermentation in CSL media is shown in figure 3b. Consumption of syngas was observed until 288 h, after which there was no consumption of syngas. The treatment that contained 10 g L-1 DTT consumed the least amount of syngas and hence produced the least amount of cells (fig. 1b). Since the gas consumption in the CSL medium with 10 g L-1 DTT ceased at around 192 h, the cell concentration also rapidly declined in that treatment.

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(a)

= = = = =

0 g L -1 2.5 g L -1 5 g L -1 7.5 g L -1 10 g L -1

= = = = =

0 g L -1 2.5 g L -1 5 g L -1 7.5 g L -1 10 g L -1

(b)

Figure 3. Pressure profiles during syngas fermentation in (a) 0.1% YE media and (b) 1% CSL media containing various concentrations of dithiothreitol (0, 2.5, 5, 7.5, and 10 g L-1). Error bars show typical experimental uncertainty.

Product Profiles Acetic acid and ethanol were the main products found in both YE and CSL media. Butanol was also produced during the fermentation process in both types of media. However, the concentration of butanol was below 0.1 g L-1 in the YE media, which is insignificant compared to the two main products (i.e., ethanol and acetic acid). Acetic acid is a primary metabolite, and its production is associated with cell growth. Hence, there was production of acetic acid until 216 h in the YE media that contained 0, 7.5, 3(1): 19-35

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and 10 g L-1 DTT (fig. 4a). However, acetic acid production was only noticed in the first 192 h in media that contained 2.5 and 5.0 g L-1 DTT. There was larger variability in the amounts of acetic acid measured after 216 h in YE media with DTT concentrations above 5.0 g L-1. However, there was a general decrease in acetic acid concentration in all YE media after 216 h. This coincides with an apparent increase in pH in the YE media that was observed after 240 h (fig. 2a). In addition, the decrease in acetic acid corresponded with an increase in ethanol production, suggesting that acetic acid was utilized by Clostridium strain P11 for ethanol formation. A decrease in acetic acid combined with an increase in ethanol production was also observed in another syngas fermentation study using the same strain P11 and reducing agents (Panneerselvam, 2009). The production of acetic acid in CSL media was also growth related (fig. 4b). In the control treatment, the maximum acetic acid concentration was about 2 g L-1 at 144 h, after which it decreased with time. The acetic acid concentration in the control at the end of 360 h of fermentation was about 1 g L-1, implying that the cells consumed about 50% of the acetic acid present in the fermentation broth (assuming there was no acetic acid production after 144 h). In the case of treatments with DTT in CSL media, the maximum acetic acid concentrations were less than the control, and the percentages of acetic acid consumed by Clostridium strain P11 were higher. The general trend observed was that acetic acid concentration decreased with increasing DTT concentration in the fermentation media (fig. 4b). In the CSL medium with 10 g L-1 DTT, the maximum acetic acid concentration was about 0.9 g L-1 at 72 h, and the cells had consumed almost all the acetic acid by 144 h. The presence of DTT in the fermentation broth seems to have stimulated the cells to consume more acetic acid. This is probably because DTT helps in regeneration of NADH from NAD+, which in turn is directly involved in ethanol production. For production of ethanol, acetic acid could have been used as a substrate, so the consumption of acetic acid increased with increasing DTT concentrations. The acetic acid concentrations (after 48 h) in CSL media with the 7.5 and 10 g L-1 DTT treatments were significantly less than the control treatments (p < 0.05). Ethanol production in YE media with and without DTT is shown in figure 5a. It can be seen that slight amounts of ethanol ( 0.05). This indicates that the concentration of DTT in the YE medium should be above 5.0 g L-1 to substantially enhance ethanol production using Clostridium strain P11. More variability in ethanol concentrations was measured in YE media with 7.5 and 10 g L-1 DTT compared to YE media with lower DTT concentrations. There was no significant difference in ethanol production in YE media with the previous two DTT concentrations (p > 0.05). However, the amounts of etha-

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Acetic Acid (g L-1 )

(a)

= 0 g L-1 = 2.5 g L -1 = 5 g L-1 = 7.5 g L -1 = 10 g L-1

= 0 g L-1 = 2.5 g L -1 = 5 g L-1 = 7.5 g L -1 = 10 g L-1

Acetic Acid (g L -1 )

(b)

Figure 4. Kinetics of acetic acid production in (a) 0.1% YE media and (b) 1% CSL media containing various concentrations of dithiothreitol (0, 2.5, 5, 7.5, and 10 g L-1). Error bars show typical experimental uncertainty.

nol produced between 288 and 360 h in YE media with 7.5 and 10 g L-1 DTT were significantly larger than in YE media either with no DTT or with 2.5 and 5.0 g L-1 DTT (p < 0.05). C. ljungdahlii produced a maximum acetic acid and ethanol concentrations of 1.2 and 0.6 g L-1, respectively, after 100 h of fermentation in 1 g L-1 YE medium with syngas that contained 4.5 mmole of CO and 1.1 mmole H2 (Younesi et al., 2005). In the present study, about 1.8 g L-1 acetic acid (fig. 4a) and 0.2 g L-1 of ethanol (fig. 5a) 3(1): 19-35

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were produced by Clostridium strain P11 after 100 h of fermentation in the YE medium without DTT. Clostridium strain P11 was fed with about 3.3 mmole of CO and 0.8 mmole H2 every 24 h. The amounts of CO or H2 consumed by Clostridium strain P11 were not measured in this experiment; therefore, it is difficult to accurately compare the results between the two clostridia strains. However, the low ethanol production by Clostridium strain P11 in the first 100 h can be generally attributed to the formation of 50% more acetic acid with Clostridium strain P11 compared to C. ljungdahlii. The addition of DTT in YE media did not have any significant effect on ethanol production during the growth phase (figs. 1a and 5a) (p > 0.05). Its effect started after the cells entered the stationary phase, at which time acetic acid production ceased (fig. 4a). This indicates that there is no need to add DTT until the cells enter the stationary phase, which could result in reducing the amount of DTT needed to enhance ethanol production. It is hypothesized that DTT donated electrons, which were used to reduce more NAD+ to NADH. The regenerated NADH might have contributed to the increased production of ethanol in the presence of DTT. The overall effect of DTT in enhancing solventogenesis is similar to that of other reducing agents used in previous studies (Ahmed, 2006; Panneerselvam, 2009; Peguin et al., 1994; Rao and Mutharasan, 1986; Rao and Mutharasan, 1988). The ethanol yield in the YE medium with 10 g L-1 DTT was 11.42 g ethanol g-1 cell mass (table 1). However, it was only 3.48 g g-1 in the absence of DTT. The ethanol yield increased with an increase in the concentration of DTT above 5.0 g L-1. It was shown that the addition of 0.1 mM of neutral red in batch reactors during syngas fermentation in YE media with C. carboxidivorans P7 increased the ethanol yield from 0.05 to 0.2 g g-1 (Ahmed, 2006). Results from another study using Clostridium strain P11 during syngas fermentation in YE media with 0.1 mM methyl viologen showed a maximum ethanol production of 1.3 g L-1 and ethanol yield of 7.5 g g-1 after 300 h, compared to 0.51 g L-1 of ethanol concentration and 2.2 g g-1 ethanol yield without the addition of methyl viologen (Panneerselvam, 2009). In the same study, the addition of 0.1 mM neutral red to the fermentation medium slightly enhanced ethanol production and yield. More ethanol production and greater yield were observed in the present study; however, the concentration of DTT used in the present study to enhance ethanol production and yield is much higher than the concentrations of other reducing agents used earlier. The profiles for ethanol production in CSL media with different DTT concentrations are shown in figure 5b. At 96 h, the ethanol concentration was 0.96 g L-1 in 7.5 g L-1 DTT, whereas the ethanol concentration was only 0.25 g L-1 in the control medium. The ethanol concentration at 216 h was similar in the CSL media with all DTT treatments (~1.3 g L-1), after which the ethanol production began to vary among the treatments. The greatest ethanol concentration observed was 2.54 g L-1 in the 5 g L-1 DTT medium at 360 h, in contrast to 1.88 g L-1 ethanol in the control treatment at 360 h. The amount of ethanol produced in the CSL medium with 5 g L-1 DTT was 35% more than in the control treatment. The concentration of ethanol at 360 h in the CSL media with the 2.5, 5, and 7.5 g L-1 DTT treatments were significantly higher than in the control (p < 0.05). The ethanol yield in the CSL medium with 10 g L-1 DTT was 16.24 g ethanol g-1 cells, which is nearly 300% greater than the ethanol yield in the CSL control treatment (5.31 g ethanol g-1 cells) (table 1). The increase in ethanol

30


= 0 g L -1 = 2.5 g L -1 = 5 g L -1 = 7.5 g L -1 = 10 g L-1

Ethanol (g L-1 )

(a)

= 0 g L -1 = 2.5 g L -1 = 5 g L -1 = 7.5 g L -1 = 10 g L -1

Ethanol (g L-1 )

(b)

Figure 5. Kinetics of ethanol production in (a) 0.1% YE media and (b) 1% CSL media containing various concentrations of dithiothreitol (0, 2.5, 5, 7.5, and 10 g L-1). Error bars show typical experimental uncertainty.

yield in CSL media cannot be considered to be an increase in ethanol productivity, as the amount of cells in the 10 g L-1 DTT treatment at 360 h was much lower than the amount of cell mass in the control treatment. The profiles for butanol production in CSL media are shown in figure 6. More butanol production was observed in CSL media than in YE media. In CSL media, butanol concentration was about 0.33 g L-1 in the control at 360 h (fig. 6), but greater butanol concentrations were observed in media that had DTT. The maximum butanol

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Butanol (g L-1 )

= 0 g L-1 = 2.5 g L -1 = 5 g L-1 = 7.5 g L -1 = 10 g L-1

Figure 6. Kinetics of butanol production in 1% CSL media containing various concentrations of dithiothreitol (0, 2.5, 5, 7.5, and 10 g L-1). Error bars show typical experimental uncertainty.

concentration observed was 0.79 g L-1 in the CSL medium with 2.5 g L-1 DTT after 360 h of fermentation, which is nearly 240% more butanol than what was produced in the control treatment (0.33 g L-1 after 360 h). In contrast, DTT had no effect on butanol production in the YE media. In CSL media, the reducing power from DTT was probably used for the production of butanol, which led to a 140% increase in butanol production (fig. 6) and just 35% increase in ethanol production in the presence of either 2.5 or 5 g L-1 DTT (fig. 5b). In addition, unknown components in CSL may have favored the routing of reducing power from DTT for more butanol production. Production of butanol from butyrylCoA is a two-step reduction reaction involving the intermediate, butyraldehyde. The production of butanol from butyryl-CoA is similar to the production of ethanol from acetyl-CoA, and the reaction involves oxidation of two molecules of NADH to NAD+. The variation in the results in replicate treatments with identical concentration of DTT (figs. 1 to 6) is indicated by error bars that show the typical experimental uncertainty. In addition to measurement errors, other possible sources for the uncertainty in the results include microbial heterogeneity of individual cells and variations in cells’ physiological state (cell-cycle stage and age), which can cause cells to grow and produce metabolites at different rates (Avery, 2006). Similar variations in the experimental data were reported using Clostridium carboxidivorans (Ahmad et al., 2006; Hurst and Lewis, 2010) and Clostridium strain P11 (Panneerselvam, 2009; Kundiyana et al., 2010). In addition, Clostridium spp. are known to have a wide variation in product yields from batch to batch, which is attributed to slight differences in culture conditions and pre-culture (Taconi et al., 2009). This is why ANOVA and statistical analysis were used in the present work to test the differences between treatments with various DTT concentrations.

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Ethanol (g L-1 )

Figure 7. Amount of ethanol produced after 360 h of fermentation in 0.1% YE medium and in 1% CSL (DTT 0 and 10 g L-1).

The effect of DTT on ethanol production by Clostridium strain P11 in CSL media was less significant compared to its effect in YE media (fig. 7). This could be due to the increased production of butanol in the presence of DTT in CSL media (fig. 6). The analysis of variance shows that DTT concentration and media type had a significant effect on final ethanol concentration (p < 0.05). Fermentations in CSL media generally resulted in a greater final ethanol concentration than did fermentations in YE media (fig. 5). The addition of DTT concentrations of 5.0, 7.5, and 10.0 g L-1 to YE media resulted in more ethanol production compared to the control (fig. 5a) (p < 0.05). The interaction between DTT concentration and media type was not significant (p > 0.05). In addition, DTT concentration, media type, and the interaction between these variables did not have a significant effect on final acetate concentrations, as shown in figure 4 (p > 0.05). The addition of DTT when cells enter the stationary phase could allow decreasing the amount of this reducing agent to effect ethanol production, which warrants further investigation.

Conclusion The effect of the reducing agent DTT on ethanol and acetic acid production by Clostridium strain P11 (ATCC PTA-7826) using simulated biomass-based syngas in two different fermentation media was investigated. Various concentrations of DTT (0 to 10.0 g L-1) were examined. The addition of DTT increased ethanol concentration in fermentations that had 0.1% yeast extract as a media nutrient, whereas DTT increased both ethanol and butanol concentrations in fermentations that had 1% CSL as a media nutrient. Addition of 7.5 g L-1 or higher concentration of DTT increased ethanol concentration in 0.1% YE media, whereas addition of 7.5 g L-1 or lower concentration of DTT increased ethanol concentration in 1% CSL media. A 350% or greater increase in ethanol concentration was observed in 0.1% YE media that contained at 3(1): 19-35

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least 7.5 g L-1 of DTT after 360 h of fermentation compared to the control medium (without DTT). There was about a 35% increase in ethanol concentration with the addition of 5 g L-1 DTT to the 1% CSL medium. Acknowledgements Support for this research was provided by USDA-CSREES Special Research Grant Award 2006-34447-16939, Oklahoma Agricultural Experiment Station, and the Oklahoma Bioenergy Center.

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