- Email: [email protected]
Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 Fuel xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Fuel journal homepage: www.elsevier.com/locate/fuel 5 6
4
Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions
7
Najeeb Kaid Nasser Al-Shorgani a,c, Mohd Sahaid Kalil b, Wan Mohtar Wan Yusoff a, Aidil Abdul Hamid a,⇑
3
8 9 10
a
School of Bioscience and Biotechnology, Faculty of Sciences and Technology, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia Department of Chemical and Process Engineering, Faculty of Engineering, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia c Department of Applied Microbiology, Faculty of Applied Sciences, Taiz University, 6803 Taiz, Yemen b
11 12 1 4 15
h i g h l i g h t s Introducing a new aerotolerant strain of Clostridium acetobutylicum YM1.
16 17
Strain YM1 is capable of producing 12.18 g/L biobutanol under aerobic conditions from 50 g/L glucose.
18
Addition of reducing chemicals improved the production of biobutanol under aerobic conditions.
19 20
Supplementation the culture with 0.01 g/L potassium ferricyanide resulted in 12.94 g/L biobutanol. YM1 was found to be able to produce catalase, SOD and NADH/NADPH oxidases.
21
a r t i c l e 2 3 3 5 24 25 26 27 28 29 30 31 32 33 34
i n f o
Article history: Received 23 December 2014 Received in revised form 28 May 2015 Accepted 30 May 2015 Available online xxxx Keywords: Aerobic fermentation Clostridium acetobutylicum YM1 Biobutanol Batch fermentation
a b s t r a c t A newly isolated strain of Clostridium acetobutylicum YM1 has a unique property of producing biobutanol from glucose under aerobic conditions. This strain exhibited the capability to grow and produce high concentrations of biobutanol under different concentrations of dissolved oxygen (DO). The growth profile and solvent production in a 5 L bioreactor were similar under anaerobic and aerobic conditions (100% initial DO saturation), and the final biobutanol production was 12.18 and 12.30 g/L, respectively. The addition of reducing chemical agents, without creating anaerobic conditions, to the culture of YM1 enhanced the production of biobutanol. Strain YM1 possesses different enzymes that are responsible for oxygen scavenging, such as superoxide dismutase (SOD), catalase and NADH/NADPH oxidases. This study provides a simple operation strategy for more efficient biobutanol production by using an aerotolerant strain of C. acetobutylicum YM1 without the need to flush the medium with nitrogen gas to ensure anaerobic conditions. Ó 2015 Published by Elsevier Ltd.
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52
1. Introduction
53
Solvent-producing Clostridium strains have gained more interest recently due to their ability to produce biobutanol via acetone–butanol–ethanol fermentation (ABE). Butanol is recognized as a candidate liquid biofuel substitute to gasoline due to its potential properties as a fuel. Butanol is currently recognized as a necessary alternative and renewable biofuel due to the expected
54 55 56 57 58
Abbreviations: ABE, acetone–butanol–ethanol; DO, dissolved oxygen; OD600, optical density at 600 nm; DTT, dithiothreitol; DTE, dithioerythritol; GC-FID, gas chromatography equipped with a flame ionization detector; NADH oxidase, nicotinamide adenine dinucleotide-oxidase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate-oxidase; SOD, superoxide dismutase; TYA, tryptone yeast-extract acetate medium. ⇑ Corresponding author. Tel.: +60 38921641; fax: +60 389216148. E-mail address: [email protected] (A.A. Hamid).
exhaustion of petroleum oil and increased demand for energy, as well as increasing global oil prices [1,2]. Butanol has many advantages as a biofuel. Butanol has a high-energy content; can be blended with gasoline or used directly in current car engines without modification; has low corrosiveness, thus, it can be transported through pipelines, has a high heat emission vaporization, and has a low contamination with water due to low solubility in water [3,4]. One of the major problems in ABE fermentation by Clostridium is the biobutanol toxicity. Clostridial growth and solvent production are completely inhibited in the presence of 13–15 g/L biobutanol. This inhibitory action explains the low final solvent production by solvent-producing Clostridium beyond 15 g/L of biobutanol concentration [5,6]. Developing tolerant strains of solvent-producing Clostridia by screening mutant strains that have high resistance to butanol, metabolic and cellular engineering using the systems approach is
http://dx.doi.org/10.1016/j.fuel.2015.05.073 0016-2361/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 2 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
crucial to solve the low production of solvent [7]. Moreover, an integration process of an in situ product recovery process with fermentation has been employed by previous researchers in order to overcome the solvent toxicity problem [8,9]. These recovery techniques include pervaporation, gas stripping, adsorption, liquid–liquid extraction, and reverse osmosis. Clostridium species, such as C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and C. sacharobutylicum, belong to genus Clostridium, which are typical obligate anaerobic bacteria. It has been proposed that the inability of obligate anaerobes to grow in an oxic atmosphere is due to their lack of systems for scavenging active oxygen species, such as superoxide anion and hydrogen peroxide, which have toxic effects on cell viability [10–12]. It has been reported that using Clostridium for ABE production at industrial levels could be limited due to the requirement of strict anaerobic conditions, and the availability of oxygen in ABE fermentation has a major impact [13]. Screening for new microbes that have potential and novel properties is consequently essential to improve the bioenergy industry [14,15]. Hence, isolation of an aerotolerant Clostridium strain that can produce high concentrations of biobutanol is crucial in ABE fermentation. The effect of oxygen on anaerobic C. acetobutylicum has been reported, and it was found that oxygen inhibits growth and DNA synthesis in C. acetobutylicum [16]. Unlike other Clostridium strains that are used in ABE fermentation, C. acetobutylicum YM1 can grow and produce biobutanol without the need to create strict anaerobic conditions by sparging the medium with oxygen-free nitrogen. Economic estimation of biobutanol production cost from corn as a substrate using C. beijerinckii BA101 has been reported in previous research [17]. It was found that biobutanol production costs at $0.34/kg based on $1.80 per bushel corn feedstock cost. The study exhibited that an improvement of biobutanol yield by 19% (0.42–0.50 g biobutanol per g glucose) would decrease the biobutanol price by 14.7% ($0.34/kg to 0.29/kg). If the corn feedstock price increases to $3.35/bushel, the estimated biobutanol production cost will be about $0.45/kg. The cost of utilities in biobutanol production through anaerobic ABE fermentation is one of the most important factors affecting the productivity [18]. The use of aerotolerant strain that does not require nitrogen gas reduces the cost. Our new isolate, aerotolerant, meets the need for supporting a typical industrial fermentation system that is easy to operate and cost effective. C. acetobutylicum YM1 was recently isolated from an agricultural area in Malaysia and was found to produce high concentrations of ABE and biohydrogen [19,20]. In this study, a new aerotolerant C. acetobutylicum YM1 strain, which was isolated from the local soil in Malaysia, was used for biobutanol production under aerobic conditions. This strain showed activity of enzymes that are responsible for scavenging O2, such as NADH oxidase, NADPH oxidase, catalase and super oxide dismutase (SOD).
127
2. Materials and methods
128
2.1. Microorganism
129
In this study, C. acetobutylicum YM1 (GenBank accession No. KC969670), isolated from local soil in Malaysia, was used, and the culture was maintained in 50% glycerol as a spore suspension at 30 °C. The inoculum was prepared by activating the spores in tryptone yeast extract acetate medium (TYA), which consisted of 20 g/L glucose, 6 g/L tryptone, 2 g/L yeast extract, 3 g/L ammonium acetate, 0.5 g/L KH2PO4, 0.3 g/L MgSO47H2O, and 0.01 g/L FeSO47H2O.
130 131 132 133 134 135 136
2.2. Fermentation conditions
137
The fermentation medium used in this study was TYA with 50 g/L glucose, and the conditions were as follows: inoculum size 10% (v/v), 30 °C, and initial pH 6.2. All fermentation experiments, except in bioreactor scale cases, were carried out in 100 mL serum bottles equipped with a rubber cap and crimped with an aluminum seal. Cultures in serum bottles were agitated by hand once a day in the first 48 h to maintain culture homogeneity. Anaerobic conditions were established by flushing the medium with nitrogen gas before inoculation. Aerobic conditions were established by sparging the medium with filtered-air until the desired dissolved oxygen saturation percentage was obtained. The bioreactor scale fermentation was conducted in a 5 L bioreactor (INFORS HT, Swiss) with a working volume of 3 L. Aerobic cultures were carried out without nitrogen flushing.
138
2.3. Analytical methods
152
Fermentation samples were collected and centrifuged at 1800 g for 5 min, and the supernatant was used for the analysis of solvents, acids and glucose. The analysis of solvents (acetone, butanol and ethanol) and acids (butyric acid and acetic acid) were conducted using a gas chromatography system (7890A GC-System, Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and a 30-m capillary column (Equity 1™; 30 m 0.32 mm 1.0 lm film thickness; Supelco Co, Bellefonate, PA, USA). The oven temperature was programmed to increase from 40 °C to 130 °C at a rate of 8 °C/min. The injector and detector temperatures were set at 250 °C and 280 °C, respectively. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. Growth was detected based on the optical density measured at 600 nm using a UV–Vis spectrophotometer (Shimadzu, UV mini-1240, Japan). The dissolved oxygen was measured by a dissolved oxygen meter (Mettler-Toledo, Swiss). The intracellular protein of the cells was measured according to the protocol of Bradford [21]. Glucose concentration was detected using a glucose oxidase kit [GOD, (E.C. 1.1.34), Roche Ltd., Swiss] following the manufacturer’s procedure.
153
2.4. Enzymes activity study
173
For determining the activities of the intracellular enzymes, catalase, NADH oxidase, NADPH oxidase and SOD, C. acetobutylicum YM1 was cultivated in TYA medium at 30 °C, 100 rpm for 24 h, and cell free extracts were prepared using the method described by Salleh et al. [22]. Catalase enzyme activity was determined using the Catalase Assay Kit (Sigma–Aldrich, USA) according to the manufacturer’s protocol. NADH oxidase and NADPH oxidase activities were measured by monitoring the oxidation of NADH or NADPH to NAD+ or NADP+, respectively at 340 nm. The assay mixture contained (in a volume of 1 mL): 50 mM air-saturated potassium phosphate buffer (pH 7.0), 0.15 mM NADH/NADPH and 100 lL of crude extract [23]. One unit of NADH/NADPH oxidase activity was determined as the amount of enzyme that catalyzed the oxidation of 1 lM of NADH/NADPH per min. Intracellular SOD activity was determined using the SOD Assay Kit (Sigma–Aldrich, USA). SOD activity in the cell-free extract is indicated by the SOD-mediated inhibition of the reaction between superoxide anion and formazon, which causes a brown discoloration of the reaction mixture that is quantified by measuring the absorbance at 450 nm. As the SOD concentration increases in this assay, the absorbance at 450 nm decreases. The SOD assays were conducted in 96 well microtitre plates and quantified using
174
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
139 140 141 142 143 144 145 146 147 148 149 150 151
154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 3
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
200
a Chromate plate reader (Chromate 4300, Awareness Technology Inc., FL, USA). All experiments were carried out at least two times, and all of the presented data are reproducible.
201
3. Results and discussion
202
3.1. Kinetics of biobutanol production under aerobic and anaerobic conditions in a 5 L bioreactor
205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220
Growth, pH, ABE and acid productions of C. acetobutylicum YM1 under aerobic and anaerobic conditions in a 5 L bioreactor were investigated. Aerobic fermentation was initiated with a DO of 100% by sparging the medium with sterilized air until 100% DO before inoculation. Anaerobic fermentation was initiated with a DO of 0% by flushing the medium with nitrogen before inoculation. Fig. 1(a) and (b) shows the results of the ABE fermentation of the YM1 strain under aerobic and anaerobic conditions, respectively, which were conducted in 5 L bioreactors. Both cultures, under aerobic and anaerobic conditions, grew well and produced ABE. The cultures reached the stationary phase after approximately 24 h of exponential growth, and similar biomass concentrations were achieved (Fig. 2(a)). A study conducted by Kawasaki et al. [24] investigating the effect of oxygen on growth of C. aminovalericum found that C. aminovalericum could not grow in the presence of 1% O2 in liquid medium at the start of the lag phase. This study showed that the
20
4.0
(a)
18 3.5 16
Solvent Production (g/L)
204
3.0
14 12
2.5
10 2.0
8 6
1.5
Acetone Butanol Ethanol ABE Acetic acid Butyric acid
4 1.0 2 0
0.5 0
20
40
60
80
100
120
140
160
Time (h) 20
4.0
(b)
3.5
Solvents Production (g/L)
203
C. aminovalericum strain could only grow anaerobically under a 100% N2 atmosphere. The growth profile of C. acetobutylicum YM1 under both conditions (aerobic and anaerobic) was associated with a gradual drop of pH from 6 to 4.66 (under aerobic condition) and 4.70 (under anaerobic condition) in the first 24 h due to the production of organic acids (butyric and acetic acids) during the log phase. Under both conditions, when the growth reached the stationary phase and the metabolism shifted to the solventogenic phase, the pH was increased slightly as a result of the acid reassimilation [25]. The growth profiles under both conditions were similar, and no significant difference was observed (Fig. 2(a)). Moreover, the specific growth rate of C. acetobutylicum YM1 in the first 12 h under aerobic and anaerobic conditions were 0.081 h 1 and 0.089 h 1, respectively. The butyric acid production under both aerobic and anaerobic conditions reached the maximum at 14 h as 3.46 g/L and 4.79 g/L, respectively. The production of acetone, butanol, and ethanol under aerobic conditions were started slightly late, but the final concentrations were similar to that produced under the anaerobic conditions (Fig. 2(b) and (c)). Glucose utilization was increased under anaerobic conditions compared with the aerobic conditions. However, the glucose was completely consumed in the first 48 h under both conditions. The glucose consumption rate under the aerobic conditions at 24 h was 0.84 g/L h and 0.97 g/L h under the anaerobic conditions. The maximum biobutanol production, under both aerobic and anaerobic conditions, was obtained at 120 h as 12.18 g/L and
Acids Production (g/L)
199
15
3.0
2.5 10 2.0
1.5 5
Acids Production (g/L)
198
Acetone Butanol Ethanol ABE Acetic acid Butyric acid
1.0
0.5
0 0
20
40
60
80
100
120
140
160
Time (h) Fig. 1. Profile of ABE fermentation under aerobic conditions (100% initial DO) and anaerobic conditions (0% DO) in a batch culture of C. acetobutylicum YM1: (a) ABE and acid production under aerobic conditions and (b) ABE and acid production under anaerobic conditions.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 4
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
3.5
60
(a)
2.5
40
2.0 30 OD(an-aerobic) OD(aerobic) Glucose (an-aerobic) Glucose (aerobic)
1.5 1.0
20 10
0.5 0.0 0
20
40
60
80
100
120
140
0 160
Time (h) 14
(b)
12
Butanol (g/L)
10 8 Aerobic An-aerobic
6 4 2 0 0
20
40
60
80
100
120
140
160
180
Time (h) 20
(c)
18 16 14
ABE (g/L)
265
The ability of strain YM1 to grow under continuous aeration by air (DO 100%) was investigated in a 5 L bioreactor with a working volume of 3 L. The medium was aerated overnight by air before inoculation, and the DO was maintained at a saturation level of 100% until 12 h after inoculation. No growth or change in pH, acids or solvents production was observed under continuous aeration. When the aeration was stopped and the medium was flushed with nitrogen for 2 min, the culture could revive the growth, acids and solvents production. After the generation of anaerobic conditions, the lag phase took approximately 12 h and was followed by the log phase, which indicated that this strain is aerotolerant, not aerobic. The highest growth was obtained at 48 h with a 2.54 OD600. The maximum final concentrations of biobutanol and ABE were obtained at 96 h from the inoculation (72 h after the growth started) as 10.25 and 16.38 g/L, respectively. The glucose concentration was constant in the first 12 h (under continuous aeration), and after the condition changed to anaerobic, the culture started to consume glucose even though no growth was observed in the first 10 h of glucose consumption. The glucose utilization rate after 24 h was high, and glucose reached 0 g/L in 72 h of culture cultivation (Fig. 3). In another experiment, the YM1 culture was started with anaerobic conditions (0% DO). After 8 h of the cultivation, the culture was sparged with air, and the DO was maintained at 100% DO saturation for 30 min. The data showed that exposure of the culture to the oxygen did not affect the growth or acids production (indicated by pH), and the growth was continued with a similar growth rate under anaerobic conditions (Fig. 4). O’Brien and Morries [16] found that aeration of C. acetobutylicum during the log phase for a period halted the growth and acids production, but the culture could regrow and produce acids after anaerobic conditions were re-established. This result is not in agreement with our study, in which strain YM1 continued to grow and produce acids when exposed to air after 6 h of growth. This study showed that strain YM1 was not affected by aeration after the growth was initiated or initial aerobic conditions (initial DO 100%), but our strain cannot grow under fully continuous aeration. Thus, our strain YM1 is aerotolerant but not fully anaerobic.
267
3.3. Profile of dissolved oxygen during the aerobic fermentation in a 5 L bioreactor
305
The data in this study showed that C. acetobutylicum YM1 is capable of growing and producing biobutanol efficiently with a high initial dissolved oxygen saturation. To study the profile of DO consumption by C. acetobutylicum YM1, a 5 L bioreactor supplemented with a dissolved oxygen meter (Mettler-Toledo, Swiss) was aerated before inoculation until the DO reached 100%. The bioreactor was inoculated by a fresh inoculum of C. acetobutylicum YM1 strain. Immediately after the inoculation, the DO in the medium was dropped gradually with time from 100% DO to 2% DO over 90 min (Fig. 5). The results showed rapid consumption of DO during the growth of strain YM1, and the growth reached 0.913 OD at 600 nm. The increase in growth was 0.067 OD during the 90 min while the decrease in pH was 0.01 at the same time. This mean that even under the presence of oxygen (100% DO saturation), the bacteria was still in the log phase; the bacteria could grow and produce acids as indicated by the drop in pH. Comparing to the growth under anaerobic conditions, the increase in growth and decrease in pH in the first 90 min were similar with a 0.07 increase in OD and 0.1 decrease in pH, respectively. The comparison indicated that the presence of high DO saturation
307
266
50
Glucose (g/L)
Growth (OD 600nm)
3.0
3.2. Effect of continuous aeration on the growth of YM1 and the solvents production
12 10 8
Aerobic An-aerobic
6 4 2
268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
0 0
20
40
60
80
100
120
140
160
180
Time (h) Fig. 2. Profiles of ABE fermentation in a batch culture of YM1 strain under aerobic and anaerobic conditions: (a) growth and glucose consumption, (b) profile of biobutanol production and (c) total ABE production profile.
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264
12.30 g/L, respectively. The maximum ABE produced from aerobic fermentation of YM1 was 18.09 g/L and was comparable to that produced from anaerobic fermentation (18.49 g/L). The production rate of biobutanol was very high with close values during the first 51 h under aerobic and anaerobic conditions as 0.24 and 0.24 g/L h, respectively; then, the biobutanol generation slowed (Fig. 2(b)). Similarly, ABE production was also high in terms of productivity in the first 51 h, and similar values were obtained under aerobic and anaerobic conditions as 0.36 g/L h and 0.36 g/L h, respectively (Fig. 2(c)). The experimental data of this study showed that the kinetics of growth and solvent production by strain YM1 were similar under anaerobic conditions, which were established by sparging the medium with nitrogen gas until the DO reached 0%, and the initial aerobic condition, which was established by sparging the medium with sterilized air until the DO reached 100%.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
306
308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 5
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
Fig. 3. Effect of continuous aeration on the growth of C. acetobutylicum YM1 and ABE production.
3.0 Aerobic
120
5.5
100 Desolved Oxygen (D.O)
5.0
1.5
4.5 Growth
1.0
80
4.0
pH
D.O (%)
2.0
pH
Growth (OD600 nm)
2.5
6.0
60
40
3.5
0.5
20
3.0
0.0 0
10
20
30
40
50
60
Time (h) Fig. 4. Effect of aerobic conditions at 8 h for 30 min on the growth of C. acetobutylicum YM1.
0 0
20
40
60
80
100
120
Time (min) Fig. 5. The profile of DO in the medium of the YM1 culture during the aerobic fermentation in a 5 L bioreactor.
327 328
329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347
in the medium did not affect the log phase, growth or acid production of the YM1 culture. 3.4. Effect of headspace on biobutanol production under aerobic conditions In this study, the effect of headspace on the growth of C. acetobutylicum YM1 and production of solvent was studied under aerobic conditions (no nitrogen was sparged). The experiments were conducted in 100 mL serum bottles, and the headspace ranges studied were 0%, 25%, 50%, 75%. The inoculum size of all the experiments was 10% (v/v), and the bottles were incubated at 30 °C for 96 h without shaking. Fig. 6(a) shows the effect of headspace on growth and solvent production. The results show that an increase in the headspace led to enhanced growth; the highest growth was found when the headspace was 75%. This can be attributed to the gas pressure in the reactor due to the production of hydrogen and CO2 in the acidogenic phase. A larger headspace allows for a decrease in the pressure of the gas and the stress on the bacterial cells. A similar observation was observed with the production of ABE. There is a correlation between ABE production and the headspace of the reactor; the biggest headspace produced the highest ABE and biobutanol of 15.75 and 10.56 g/L, respectively.
Head space also affects the biobutanol to acetone ratio, and the results showed that an increase of headspace results in a decrease of the biobutanol to acetone ratio (Fig. 6(b)). At a headspace of 0%, the B: A ratio was 3.44 while at a headspace of 75%, the B: A ratio was 2.47. The effect of gas pressure on ABE fermentation was reported by Woods and Jones [26]. It was stated that ABE fermentation under a pressure of 2000 kPa resulted in an increase in the yield of biobutanol while the butyrate yield was decreased. The yield of biobutanol and ethanol but not acetone was reported to be enhanced by increasing the headspace pressure from 100 to 250 kPa [26]. It is clear that the headspace pressure has a significant effect and increasing the headspace pressure in the reactor vessel led to enhanced biobutanol production, which can be attributed to the increase of dissolved hydrogen in the fermentation medium, thus affecting the production of ABE [27]. Under high pressure, C. acetobutylicum produces less primary metabolites (H2 and acids) and increases the solvent as secondary metabolites. Headspace pressure in the fermentation vessel as a stress factor can change the bacterial metabolites and affect the ratio of ABE, as well as force the bacteria to shift from acidogenic phase to solventogenic phase.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
Growth (OD600) & Solvent Producon (g/L)
6
12
(a)
10
Growth OD Acetone Butanol Ethanol
8 6 4 2 0 0
25
50
75
Head space (%) 4
(b)
B:A rao
3
2
1
saturated by oxygen). It was found that C. acetobutylicum YM1 could grow and produce biobutanol under different DO concentrations. The results showed that increasing the DO in the medium resulted in a decrease of biobutanol production. The data showed that this strain can tolerate the presence of oxygen (DO) in the medium, and it was able to grow and produce ABE even under an initial DO of 100%. As illustrated in Table 1, the C. acetobutylicum YM1 strain growth was similar, and no significant differences in growth were seen even under high DO percentages. The results showed an inverse correlation between DO and biobutanol production (Table 1).The biobutanol production under anaerobic conditions (0% DO) was 11.18 g/L while biobutanol production under 100% DO was 7.89 g/L. In terms of biobutanol yield, the highest biobutanol yield of 0.224 was obtained from 0% DO; a DO of 10% and 25% showed the same yield of 0.221. Increasing the initial DO in the medium resulted in a decrease in the biobutanol yield (Table 1). The biobutanol yield was inversely proportional to DO concentration. Importantly, in a previous study by O’Brien and Morries [16], it was stated that the growth of C. acetobutylicum was ceased under full aerobic conditions. The growth also ceased during a short aeration period in the log phase, but the growth recovered after stopping the aeration with no cell damage being observed. The ability of YM1 (as any solvent-producing Clostridium) to produce gases (H2 and CO2) in the acidogenic phase can contribute to a reduction in the oxygen percentage in the culture during the growth, which trends the conditions to more anaerobic over time.
385
3.6. Effect of agitation speed on biobutanol production
412
The agitation speed under anaerobic conditions was reported to increase the dissolved hydrogen in the medium [27], while in this study agitation speed under aerobic conditions was assumed to increase the dissolved oxygen in the fermentation medium and subsequently facilitate more aerobic conditions. Hence to show the capability of the YM1 strain to grow and produce biobutanol under more aerobiosis, experiments were conducted under an initial DO of 75% in 100 mL serum bottles with a working volume of 50 mL (50% head space), and the culture was agitated after the inoculation at different agitation speeds as shown in Table 2. The results showed that no significance differences in solvents and biobutanol productions were observed when the agitation
413
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
0 0
25
50
75
Head space (%) Fig. 6. Effect of headspace on the growth and ABE production under aerobic conditions by C. acetobutylicum YM1: (a) growth and ABE profile and (b) biobutanol to acetone (B:A) ratio.
370 371 372 373 374 375 376
It was found that during ABE fermentation, pressures of up to 105 kPa can be generated naturally due to the elevation of gas formation during the fermentation. Maintenance of positive head space pressure during the fermentation at 105 kPa led to enhanced biobutanol productivity, and the enhancement in biobutanol production was 60% greater compared to non-pressurized cultures [28].
377
3.5. Effect of dissolved oxygen on biobutanol production
378
To study the effect of dissolved oxygen on the growth of C. acetobutylicum YM1 and biobutanol production, various dissolved oxygen percentages were applied to the fermentation medium in sealed serum bottles before autoclaving by sparging filtered air through an air filter (0.2 l pore size) or nitrogen gas (95% N2 and 5% O2) to obtain the desired dissolved oxygen. The DO concentrations were varied from 0% (anaerobic) to 100% (fully
379 380 381 382 383 384
Table 2 Effect of agitation speed on the growth of YM1 and solvent production under aerobic conditions. Agitation speed (rpm)
Growth (OD 600 nm)
Solvent production (g/L) Acetone
Butanol
Ethanol
ABE
0 25 50 100 200
2.196 2.297 2.214 2.218 1.983
3.20 3.91 4.15 4.17 2.31
9.12 9.72 9.12 9.15 7.12
0.90 1.16 0.87 0.97 0.54
13.22 14.79 14.14 14.29 9.97
Table 1 The effect of DO on metabolites production and growth of C. acetobutylicum YM1. DO (%)
0 10 25 50 75 100
Solvent production (g/L)
Acid production (g/L)
Acetone
Butanol
Ethanol
ABE
Acetic acid
Butyric acid
4.68 3.77 3.61 3.17 3.54 3.44
11.18 10.83 10.37 8.16 8.70 7.89
1.15 1.23 1.21 0.71 0.82 0.64
17.00 15.83 15.19 12.03 13.05 11.97
5.17 4.22 4.14 4.80 3.59 4.86
1.25 1.96 1.39 2.44 1.47 1.37
Growth (OD600)
Residual glucose (g/L)
Butanol yield (g/g)
2.36 2.33 2.32 2.36 2.29 2.31
0.00 1.03 3.12 9.21 9.70 7.79
0.224 0.221 0.221 0.200 0.216 0.187
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
414 415 416 417 418 419 420 421 422 423 424
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 7
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440
441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475
speed was between 0 and 100 rpm (Table 2). When the speed was more than 100 rpm, the growth, ABE and biobutanol were decreased compared to agitations of 100 rpm and below. The bacterial cells accumulated in the bottom of the bottle when the agitation speed exceeded 100 rpm. It was reported that agitation of the culture in ABE production is not required due to the sufficient mixing, which results from the gas evolution during the fermentation [28]. This finding supported our results in which no differences were found, in terms of fermentation products, due to varying the agitation. However, our aim was to investigate the effect of agitation as an aeration method. Previous studies have shown that under anaerobic conditions of C. acetobutylicum, high levels of hydrogen supersaturation can be achieved without agitation, and the supersaturation of hydrogen leads to the production of alcohols as reduction products, such as ethanol and biobutanol [29,30]. 3.7. Improved biobutanol production by supplementing the medium with reducing agents under aerobic conditions Chemical reducing agents were added at very low concentrations to support growth and to reach the same level of biobutanol concentrations as obtained from anaerobic fermentation, as well as to avoid the need for nitrogen gas sparging as an economic consideration. To investigate the effect of reducing chemical agents supplementation on ABE fermentation under aerobic conditions, various reducing agents, including resazurine, Na2S, DTT, DTE and potassium ferricyanide, were investigated in a series of experiments with different concentrations (data not shown). Table 3 illustrates the optimized concentrations of each reducing agent, production of ABE and acids, as well as the biobutanol yield. In the control culture of YM1, which was conducted under aerobic conditions without any supplementation, the culture produced 13.89 g/L ABE and 9.96 g/L biobutanol, and the biobutanol yield was 0.199 g/g. Meanwhile, batch fermentation of the YM1 culture under anaerobic conditions resulted in production of 14.62 g/L ABE and 10.61 g/L biobutanol with a biobutanol yield of 0.21 g/L. The increase in biobutanol production under anaerobic conditions was 6.52% compared to the aerobic conditions. Table 3 shows that all of the reducing agents used in this study improved the production of biobutanol and ABE, and the amount produced was higher than that produced under anaerobic conditions. When the culture was supplemented with 0.01 g/L potassium ferricyanide, the production of ABE and biobutanol was 17.54 and 12.94 g/L, respectively, with a biobutanol yield of 0.259 g/g. The yield of biobutanol was increased from 0.199 in the control culture to 0.259 with the addition of 0.01 g/L potassium ferricyanide (30.15%). Potassium ferricyanide is usually used as a physiological buffer to increase the solution’s redox potential, maintain the oxidation–reduction potential and create microaerobic
conditions. The addition of potassium ferricyanide resulted in a cultural conditions less sensitive to oxygen. This is in agreement with that reported by Walden and Hentges [31] who employed C. perfringens. The addition of 0.001 g/L resazurine to the medium resulted in production of 11.17 g/L biobutanol and 15.31 g/L ABE with a biobutanol yield of 0.223 g/g. However, the cell growth was decreased compared to the control culture, which can be attributed to the toxic effect of resazurine on the growth of YM1. DTT and DTE also proved to be good supplementary agents that could enhance the production of biobutanol to 11.60 g/L and 12.40 g/L, as well as improve the growth of YM1 under aerobic conditions (Table 3). Supplementation of the medium with 0.05 g/L Na2S contributed to an increase in ABE and biobutanol production by YM1 under aerobic conditions, and the production of ABE and biobutanol were 17.11 and 12.19 g/L, respectively. The yield of biobutanol was 0.224 g/g, and the cell growth was enhanced 15% compared to the control culture. Hence, it was found that the addition of reducing agents to the fermentation medium under aerobic conditions scavenged the DO and subsequently, improved the biobutanol production to levels higher than that produced under the anaerobic conditions. The results indicate that supplementation with reducing chemical agents at very low concentrations improved the biobutanol and ABE production even under aerobic conditions, and this information can be used to reduce the cost of sparging nitrogen during the ABE fermentation to facilitate anaerobic conditions.
476
3.8. NADH /NADPH oxidase, catalase and superoxide dismutase (SOD) enzyme activity
503
Solvent-producing Clostridium species were reported as non-catalase producers, which is an important enzyme for scavenging the hydrogen peroxide produced via the O2-reduction system [24,32]. Under aerobic conditions, the bacterial growth response to O2 is related to the capability of strains to induce superoxide dismutase (SOD) and NADH/NADPH oxidase enzymes [33]. NADH oxidase and NADPH oxidase activates were investigated as scavengers for hydrogen peroxide in cell-free extracts of the YM1 strain. The results revealed that NADH oxidase and NADPH oxidase showed activities. The activity of NADH oxidase was 49.64 U/mg protein, and the activity of NADPH oxidase was 19.95 U/mg protein (Table 4). These results showed that the NADH/NADPH oxidation system is involved in the O2-reduction system in C. acetobutylicum YM1. Strain YM1 also showed activity of SOD in cell crude extracts, and the SOD activity was 59.02% (% inhibition). Moreover, catalase enzyme was detected in the cell crude extract of this strain, and the catalase activity was 61.19 U/mg protein (Table 4). Brioukhanov et al. reported that most of the investigated Clostridium species and acetogens were negative for catalase enzymes [34]. In contrast, Gaenko et al. reported that some clostridia had catalase enzyme
505
Table 3 Impact of reducing agents in the fermentation of ABE by the YM1 strain under aerobic conditions in flask scale. Reducing agent
Resazurine Na2S DTT DTE Potassium ferricyanide Control (aerobic) Control (anaerobic)
Concentration (g/L)
0.001 0.05 0.3 0.3 0.01
Growth (OD600 nm)
1.87 2.53 2.252 2.207 2.360 2.200 2.251
Solvents (g/L)
Acids (g/L)
Butanol yield (YP/S)
Acetone
Butanol
Ethanol
ABE
Acetic
Butyric
3.11 3.79 3.94 4.24 3.51 3.02 2.92
11.17 12.19 11.60 12.40 12.94 9.96 10.61
1.02 1.12 1.13 1.11 1.09 0.91 1.09
15.31 17.11 16.67 17.75 17.54 13.89 14.62
0.86 0.59 1.12 1.24 1.54 1.01 0.87
0.53 0.57 1.51 1.27 1.32 1.07 0.71
0.223 0.244 0.232 0.248 0.259 0.199 0.210
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502
504
506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 8
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
Table 4 Oxygen scavenging enzyme activity in a cell-free extract of YM1 grown under initial 50% DO. Enzyme
Activity
SOD activity (%) Catalase (U/mg Protein) NADH Oxidase (U/mg Protein) NADPH Oxidase (U/mg Protein)
59.02 61.19 49.64 19.95
573
activity [35]. Our strain YM1 exhibited the activity of a catalase enzyme, which is in agreement with Gaenko et al. [35] and not in agreement with Brioukhanov et al. [34]. The data in this study indicated that this strain possesses some enzymes, such as catalase, SOD, NADH oxidase and NADPH oxidase, which are important to protect the bacterial cells against the toxic effect of activated species of oxygen, such as superoxide anion and hydrogen peroxide. These enzymes may be the reason why this strain can tolerate the presence of oxygen. The enzymes responsible for scavenging O2 in anaerobic bacteria typically are NADH oxidase and NADPH oxidase. These enzymes can scavenge O2 to produce water or hydrogen peroxide from a dioxygen reaction [24,36]. C. acetobutylicum, which is an obligatory anaerobic, gram-positive acetone–butanol–ethanol producer, can tolerate air exposure due to its ability to activate its oxidative stress response leading to subsequent rapid cellular O2 consumption and high NADH/NADPH oxidase activity in crude extracts [37]. It was found that the increase in NADH oxidase activity was due to the exposure of C. acetobutylicum to oxygen and was not due to the rise in the culture’s redox potential (Eh) [16]. Aerobic and facultative bacteria have highly regulated antioxidative enzymes, such as catalase and superoxide dismutase. These enzymes decrease the oxygen toxicity and mutagenic effects to maintain the cellular concentration of oxidative metabolites at low levels. Strictly anaerobic bacteria lack the antioxidative enzymes and thus are incapable of surviving oxidative stress [38]. Exposure of C. acetobutylicum to oxygen for short times after the bacteria reached the exponential phase resulted in halted growth, decreased glucose utilization, and inhibited the synthesis of protein, DNA and RNA [16]. At oxygen concentrations of 40 lM, the growth of C. acetobutylicum was inhibited even in a medium composed with dithiothreitol at 50 mV [16]. In the presence of oxygen, only the SOD possessing microorganisms can survive while the catalase enzyme is not so important [39]. Previous studies also revealed that the microorganisms tolerance to oxygen is related to SOD activity, and the higher aerotolerant microorganisms showed higher SOD activity than microorganisms with low SOD activity or microorganisms lacking SOD [40]. In black-pigmented Bacteroides, superoxide dismutase activity exhibited a higher correlation with oxygen tolerance than with the activity of NADH oxidase [41]. However, microorganisms can obviously contain significant superoxide dismutase activity and still be incapable of growth in air. Hence, the presence of superoxide dismutase enzyme is not restricted to microorganisms capable of growth in air [39].
574
4. Conclusion
575
The ability of C. acetobutylicum YM1 strain to grow and produce high concentrations of biobutanol efficiently under conditions that are not fully anaerobic deserves interest, as it has been stated by previous studies that solvent-producing Clostridium strains are obligatory anaerobic. This strain possesses an oxygen scavenging system that is represented by SOD, catalase and NADH/NADPH
527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572
576 577 578 579 580
oxidases. The growth profile and solvent production were similar when the culture was cultivated under aerobic and anaerobic conditions. The addition of small concentrations of reducing agents resulted in a considerable improvement in biobutanol production, as well as a substitute for using anaerobic conditions.
581
Acknowledgment
586
The authors would like to thank University Kebangsaan Malaysia for funding this study under grants; DLP-2013-023, GUP-2013-037, UKM-DIP-2012-30 and UKM-DLP-2012-007.
587
References
590
[1] Kheyrandish M, Asadollahi MA, Jeihanipour A, Doostmohammadi M, RismaniYazdi H, Karimi K. Direct production of acetone–butanol–ethanol from waste starch by free and immobilized Clostridium acetobutylicum. Fuel 2015;142:129–33. [2] Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS. Fermentative butanol production by clostridia. Biotechnol Bioeng 2008;101:209–28. [3] Khanna S, Goyal A, Moholkar VS. Production of n-butanol from biodiesel derived crude glycerol using Clostridium pasteurianum immobilized on Amberlite. Fuel 2013;112:557–61. [4] Al-Shorgani NKN, Ali E, Kalil MS, Yusoff WMW. Bioconversion of butyric acid to butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a limited nutrient medium. Bioenergy Res 2012;5:287–93. [5] Al-Shorgani NKN, Kalil MS, Yusoff WMW. The effect of different carbon sources on biobutanol production using Clostridium saccharoperbutylacetonicum N14. Biotechnology 2011;10:280–5. [6] Chen B-Y, Chuang F-Y, Lin C-L, Chang J-S. Deciphering butanol inhibition to Clostridial species in acclimatized sludge for improving biobutanol production. Biochem Eng J 2012;69:100–5. [7] Lee SY, Lee DY, Kim TY. Systems biotechnology for strain improvement. Trends Biotechnol 2005;23:349–58. [8] Lee S, Park J, Jang S, Nielsen L, Kim J, Jung K. Fermentative butanol production by clostridia. Biotechnol Bioeng 2008;101:209–28. [9] Qureshi N, Blaschek HP. Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation. Appl Biochem Biotechnol 2000;84–86:225–35. [10] Andreesen J, Bahl H, Gottschalk G. Introduction to the physiology and biochemistry of the genus Clostridium. In: Minton N, Clarke D, editors. Clostridia, vol. 3. US: Springer; 1989. p. 27–62. [11] Jones DT, Woods DR. Acetone-butanol fermentation revisited. Microbiol Rev 1986;50:484–524. [12] Kawasaki S, Nakagawa T, Nishiyama Y, Benno Y, Uchimura T, Komagata K, et al. Effect of oxygen on the growth of Clostridium butyricum (type species of the genus Clostridium), and the distribution of enzymes for oxygen and for active oxygen species in Clostridia. J Ferment Bioeng 1998;86:368–72. [13] Yazdani SS, Gonzalez R. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 2007;18:213–9. [14] Bramono SE, Lam YS, Ong SL, He J. A mesophilic Clostridium species that produces butanol from monosaccharides and hydrogen from polysaccharides. Bioresour Technol 2011;102:9558–63. [15] Chua TK, Liang D-W, Qi C, Yang K-L, He J. Characterization of a butanol– acetone-producing Clostridium strain and identification of its solventogenic genes. Bioresour Technol 2013;135:372–8. [16] O’Brien RW, Morris JG. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol 1971;68:307–18. [17] Qureshi N, Blaschek HP. ABE production from corn: a recent economic evaluation. J Ind Microbiol Biotechnol 2001;27:292–7. [18] Qureshi N, Saha BC, Cotta MA, Singh V. An economic evaluation of biological conversion of wheat straw to butanol: a biofuel. Energy Convers Manage 2013;65:456–62. [19] Abdeshahian P, Al-Shorgani NKN, Salih NKM, Shukor H, Kadier A, Hamid AA, et al. The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process. Int J Hydrogen Energy 2014;39:12524–31. [20] Al-Shorgani NKN, Hamid AA, Yusoff WMW, Kalil MS. Pre-optimization of medium for biobutanol production by a new isolate of solvent-producing Clostridium. BioResources 2013;8:1420–30. [21] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [22] Salleh M, Tsuey L, Ariff A. The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 utilizing different pH control strategies. Biotechnol Bioprocess Eng 2008;13:33–9. [23] Kawasaki S, Watamura Y, Ono M, Watanabe T, Takeda K, Niimura Y. Adaptive responses to oxygen stress in obligatory anaerobes Clostridium acetobutylicum and Clostridium aminovalericum. Appl Environ Microbiol 2005;71:8442–50. [24] Kawasaki S, Ishikura J, Chiba D, Nishino T, Niimura Y. Purification and characterization of an H2O-forming NADH oxidase from Clostridium
591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
582 583 584 585
588 589
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682
[25]
[26] [27] [28] [29]
[30]
[31]
[32]
aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria. Arch Microbiol 2004;181:324–30. Kumar M, Gayen K, Saini S. Role of extracellular cues to trigger the metabolic phase shifting from acidogenesis to solventogenesis in Clostridium acetobutylicum. Bioresour Technol 2013;138:55–62. Woods DR, Jones DT. Physiological responses of Bacteroides and Clostridium strains to environmental stress factors. Adv Microb Physiol 1986;28:1–64. Doremus MG, Linden JC, Moreira AR. Agitation and pressure effects on acetone–butanol fermentation. Biotechnol Bioeng 1985;27:852–60. Maddox IS. The acetone–butanol–ethanol fermentation: recent progress in technology. Biotechnol Genet Eng Rev 1989;7:189–220. Lamed R, Lobos J, Su T. Effects of stirring and hydrogen on fermentation products of Clostridium thermocellum. Appl Environ Microbiol 1988;54:1216–21. Lobos J, Lamed RJ, Su TM. H2 supersaturation: its effect on end product formation in anaerobic fermentations. Annual Meeting of the American Society for Microbiology. Atlanta; 1982. p. 198. Walden WC, Hentges DJ. Differential effects of oxygen and oxidation reduction potential on the multiplication of three species of anaerobic intestinal bacteria. Appl Microbiol 1975;30:781–5. Keis S, Shaheen R, Jones DT. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov. Int J Syst Evol Microbiol 2001;51:2095–103.
9
[33] Higuchi M, Shimada M, Yamamoto Y, Hayashi T, Koga T, Kamio Y. Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans. J Gen Microbiol 1993;139:2343–51. [34] Brioukhanov AL, Thauer RK, Netrusov AI. Catalase and superoxide dismutase in the cells of strictly anaerobic microorganisms. Microbiology 2002;71:281–5. [35] Gaenko GP, Reshetnikova IV, Duda VI, Plekhanova IO, Gusev MV. Superoxide dismutase in Clostridium butyricum spores. Mikrobiologiia 1985;54:322–4. [36] Reed DW, Millstein J, Hartzell PL. H2O2-forming NADH oxidase with diaphorase (cytochrome) activity from Archaeoglobus fulgidus. J Bacteriol 2001;183:7007–16. [37] Hillmann F, Fischer R-J, Saint-Prix F, Girbal L, Bahl H. PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum. Mol Microbiol 2008;68:848–60. [38] Brioukhanov AL, Netrusov AI. Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review. Appl Biochem Microbiol 2007;43:567–82. [39] Hewitt J, Morris JG. Superoxide dismutase in some obligately anaerobic bacteria. FEBS Lett 1975;50:315–8. [40] Privalle CT, Gregory EM. Superoxide dismutase and O2 lethality in Bacteroides fragilis. J Bacteriol 1979;138:139–45. [41] Amano A, Tamagawa H, Takagaki M, Murakami Y, Shizukuishi S, Tsunemitsu A. Relationship between enzyme activities involved in oxygen metabolism and oxygen tolerance in black-pigmented bacteroides. J Dent Res 1988;67:1196–9.
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
No. of Pages 9, Model 5G
6 June 2015 Fuel xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Fuel journal homepage: www.elsevier.com/locate/fuel 5 6
4
Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions
7
Najeeb Kaid Nasser Al-Shorgani a,c, Mohd Sahaid Kalil b, Wan Mohtar Wan Yusoff a, Aidil Abdul Hamid a,⇑
3
8 9 10
a
School of Bioscience and Biotechnology, Faculty of Sciences and Technology, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia Department of Chemical and Process Engineering, Faculty of Engineering, Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia c Department of Applied Microbiology, Faculty of Applied Sciences, Taiz University, 6803 Taiz, Yemen b
11 12 1 4 15
h i g h l i g h t s Introducing a new aerotolerant strain of Clostridium acetobutylicum YM1.
16 17
Strain YM1 is capable of producing 12.18 g/L biobutanol under aerobic conditions from 50 g/L glucose.
18
Addition of reducing chemicals improved the production of biobutanol under aerobic conditions.
19 20
Supplementation the culture with 0.01 g/L potassium ferricyanide resulted in 12.94 g/L biobutanol. YM1 was found to be able to produce catalase, SOD and NADH/NADPH oxidases.
21
a r t i c l e 2 3 3 5 24 25 26 27 28 29 30 31 32 33 34
i n f o
Article history: Received 23 December 2014 Received in revised form 28 May 2015 Accepted 30 May 2015 Available online xxxx Keywords: Aerobic fermentation Clostridium acetobutylicum YM1 Biobutanol Batch fermentation
a b s t r a c t A newly isolated strain of Clostridium acetobutylicum YM1 has a unique property of producing biobutanol from glucose under aerobic conditions. This strain exhibited the capability to grow and produce high concentrations of biobutanol under different concentrations of dissolved oxygen (DO). The growth profile and solvent production in a 5 L bioreactor were similar under anaerobic and aerobic conditions (100% initial DO saturation), and the final biobutanol production was 12.18 and 12.30 g/L, respectively. The addition of reducing chemical agents, without creating anaerobic conditions, to the culture of YM1 enhanced the production of biobutanol. Strain YM1 possesses different enzymes that are responsible for oxygen scavenging, such as superoxide dismutase (SOD), catalase and NADH/NADPH oxidases. This study provides a simple operation strategy for more efficient biobutanol production by using an aerotolerant strain of C. acetobutylicum YM1 without the need to flush the medium with nitrogen gas to ensure anaerobic conditions. Ó 2015 Published by Elsevier Ltd.
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52
1. Introduction
53
Solvent-producing Clostridium strains have gained more interest recently due to their ability to produce biobutanol via acetone–butanol–ethanol fermentation (ABE). Butanol is recognized as a candidate liquid biofuel substitute to gasoline due to its potential properties as a fuel. Butanol is currently recognized as a necessary alternative and renewable biofuel due to the expected
54 55 56 57 58
Abbreviations: ABE, acetone–butanol–ethanol; DO, dissolved oxygen; OD600, optical density at 600 nm; DTT, dithiothreitol; DTE, dithioerythritol; GC-FID, gas chromatography equipped with a flame ionization detector; NADH oxidase, nicotinamide adenine dinucleotide-oxidase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate-oxidase; SOD, superoxide dismutase; TYA, tryptone yeast-extract acetate medium. ⇑ Corresponding author. Tel.: +60 38921641; fax: +60 389216148. E-mail address: [email protected] (A.A. Hamid).
exhaustion of petroleum oil and increased demand for energy, as well as increasing global oil prices [1,2]. Butanol has many advantages as a biofuel. Butanol has a high-energy content; can be blended with gasoline or used directly in current car engines without modification; has low corrosiveness, thus, it can be transported through pipelines, has a high heat emission vaporization, and has a low contamination with water due to low solubility in water [3,4]. One of the major problems in ABE fermentation by Clostridium is the biobutanol toxicity. Clostridial growth and solvent production are completely inhibited in the presence of 13–15 g/L biobutanol. This inhibitory action explains the low final solvent production by solvent-producing Clostridium beyond 15 g/L of biobutanol concentration [5,6]. Developing tolerant strains of solvent-producing Clostridia by screening mutant strains that have high resistance to butanol, metabolic and cellular engineering using the systems approach is
http://dx.doi.org/10.1016/j.fuel.2015.05.073 0016-2361/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 2 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
crucial to solve the low production of solvent [7]. Moreover, an integration process of an in situ product recovery process with fermentation has been employed by previous researchers in order to overcome the solvent toxicity problem [8,9]. These recovery techniques include pervaporation, gas stripping, adsorption, liquid–liquid extraction, and reverse osmosis. Clostridium species, such as C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and C. sacharobutylicum, belong to genus Clostridium, which are typical obligate anaerobic bacteria. It has been proposed that the inability of obligate anaerobes to grow in an oxic atmosphere is due to their lack of systems for scavenging active oxygen species, such as superoxide anion and hydrogen peroxide, which have toxic effects on cell viability [10–12]. It has been reported that using Clostridium for ABE production at industrial levels could be limited due to the requirement of strict anaerobic conditions, and the availability of oxygen in ABE fermentation has a major impact [13]. Screening for new microbes that have potential and novel properties is consequently essential to improve the bioenergy industry [14,15]. Hence, isolation of an aerotolerant Clostridium strain that can produce high concentrations of biobutanol is crucial in ABE fermentation. The effect of oxygen on anaerobic C. acetobutylicum has been reported, and it was found that oxygen inhibits growth and DNA synthesis in C. acetobutylicum [16]. Unlike other Clostridium strains that are used in ABE fermentation, C. acetobutylicum YM1 can grow and produce biobutanol without the need to create strict anaerobic conditions by sparging the medium with oxygen-free nitrogen. Economic estimation of biobutanol production cost from corn as a substrate using C. beijerinckii BA101 has been reported in previous research [17]. It was found that biobutanol production costs at $0.34/kg based on $1.80 per bushel corn feedstock cost. The study exhibited that an improvement of biobutanol yield by 19% (0.42–0.50 g biobutanol per g glucose) would decrease the biobutanol price by 14.7% ($0.34/kg to 0.29/kg). If the corn feedstock price increases to $3.35/bushel, the estimated biobutanol production cost will be about $0.45/kg. The cost of utilities in biobutanol production through anaerobic ABE fermentation is one of the most important factors affecting the productivity [18]. The use of aerotolerant strain that does not require nitrogen gas reduces the cost. Our new isolate, aerotolerant, meets the need for supporting a typical industrial fermentation system that is easy to operate and cost effective. C. acetobutylicum YM1 was recently isolated from an agricultural area in Malaysia and was found to produce high concentrations of ABE and biohydrogen [19,20]. In this study, a new aerotolerant C. acetobutylicum YM1 strain, which was isolated from the local soil in Malaysia, was used for biobutanol production under aerobic conditions. This strain showed activity of enzymes that are responsible for scavenging O2, such as NADH oxidase, NADPH oxidase, catalase and super oxide dismutase (SOD).
127
2. Materials and methods
128
2.1. Microorganism
129
In this study, C. acetobutylicum YM1 (GenBank accession No. KC969670), isolated from local soil in Malaysia, was used, and the culture was maintained in 50% glycerol as a spore suspension at 30 °C. The inoculum was prepared by activating the spores in tryptone yeast extract acetate medium (TYA), which consisted of 20 g/L glucose, 6 g/L tryptone, 2 g/L yeast extract, 3 g/L ammonium acetate, 0.5 g/L KH2PO4, 0.3 g/L MgSO47H2O, and 0.01 g/L FeSO47H2O.
130 131 132 133 134 135 136
2.2. Fermentation conditions
137
The fermentation medium used in this study was TYA with 50 g/L glucose, and the conditions were as follows: inoculum size 10% (v/v), 30 °C, and initial pH 6.2. All fermentation experiments, except in bioreactor scale cases, were carried out in 100 mL serum bottles equipped with a rubber cap and crimped with an aluminum seal. Cultures in serum bottles were agitated by hand once a day in the first 48 h to maintain culture homogeneity. Anaerobic conditions were established by flushing the medium with nitrogen gas before inoculation. Aerobic conditions were established by sparging the medium with filtered-air until the desired dissolved oxygen saturation percentage was obtained. The bioreactor scale fermentation was conducted in a 5 L bioreactor (INFORS HT, Swiss) with a working volume of 3 L. Aerobic cultures were carried out without nitrogen flushing.
138
2.3. Analytical methods
152
Fermentation samples were collected and centrifuged at 1800 g for 5 min, and the supernatant was used for the analysis of solvents, acids and glucose. The analysis of solvents (acetone, butanol and ethanol) and acids (butyric acid and acetic acid) were conducted using a gas chromatography system (7890A GC-System, Agilent Technologies, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and a 30-m capillary column (Equity 1™; 30 m 0.32 mm 1.0 lm film thickness; Supelco Co, Bellefonate, PA, USA). The oven temperature was programmed to increase from 40 °C to 130 °C at a rate of 8 °C/min. The injector and detector temperatures were set at 250 °C and 280 °C, respectively. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. Growth was detected based on the optical density measured at 600 nm using a UV–Vis spectrophotometer (Shimadzu, UV mini-1240, Japan). The dissolved oxygen was measured by a dissolved oxygen meter (Mettler-Toledo, Swiss). The intracellular protein of the cells was measured according to the protocol of Bradford [21]. Glucose concentration was detected using a glucose oxidase kit [GOD, (E.C. 1.1.34), Roche Ltd., Swiss] following the manufacturer’s procedure.
153
2.4. Enzymes activity study
173
For determining the activities of the intracellular enzymes, catalase, NADH oxidase, NADPH oxidase and SOD, C. acetobutylicum YM1 was cultivated in TYA medium at 30 °C, 100 rpm for 24 h, and cell free extracts were prepared using the method described by Salleh et al. [22]. Catalase enzyme activity was determined using the Catalase Assay Kit (Sigma–Aldrich, USA) according to the manufacturer’s protocol. NADH oxidase and NADPH oxidase activities were measured by monitoring the oxidation of NADH or NADPH to NAD+ or NADP+, respectively at 340 nm. The assay mixture contained (in a volume of 1 mL): 50 mM air-saturated potassium phosphate buffer (pH 7.0), 0.15 mM NADH/NADPH and 100 lL of crude extract [23]. One unit of NADH/NADPH oxidase activity was determined as the amount of enzyme that catalyzed the oxidation of 1 lM of NADH/NADPH per min. Intracellular SOD activity was determined using the SOD Assay Kit (Sigma–Aldrich, USA). SOD activity in the cell-free extract is indicated by the SOD-mediated inhibition of the reaction between superoxide anion and formazon, which causes a brown discoloration of the reaction mixture that is quantified by measuring the absorbance at 450 nm. As the SOD concentration increases in this assay, the absorbance at 450 nm decreases. The SOD assays were conducted in 96 well microtitre plates and quantified using
174
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
139 140 141 142 143 144 145 146 147 148 149 150 151
154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172
175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 3
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
200
a Chromate plate reader (Chromate 4300, Awareness Technology Inc., FL, USA). All experiments were carried out at least two times, and all of the presented data are reproducible.
201
3. Results and discussion
202
3.1. Kinetics of biobutanol production under aerobic and anaerobic conditions in a 5 L bioreactor
205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220
Growth, pH, ABE and acid productions of C. acetobutylicum YM1 under aerobic and anaerobic conditions in a 5 L bioreactor were investigated. Aerobic fermentation was initiated with a DO of 100% by sparging the medium with sterilized air until 100% DO before inoculation. Anaerobic fermentation was initiated with a DO of 0% by flushing the medium with nitrogen before inoculation. Fig. 1(a) and (b) shows the results of the ABE fermentation of the YM1 strain under aerobic and anaerobic conditions, respectively, which were conducted in 5 L bioreactors. Both cultures, under aerobic and anaerobic conditions, grew well and produced ABE. The cultures reached the stationary phase after approximately 24 h of exponential growth, and similar biomass concentrations were achieved (Fig. 2(a)). A study conducted by Kawasaki et al. [24] investigating the effect of oxygen on growth of C. aminovalericum found that C. aminovalericum could not grow in the presence of 1% O2 in liquid medium at the start of the lag phase. This study showed that the
20
4.0
(a)
18 3.5 16
Solvent Production (g/L)
204
3.0
14 12
2.5
10 2.0
8 6
1.5
Acetone Butanol Ethanol ABE Acetic acid Butyric acid
4 1.0 2 0
0.5 0
20
40
60
80
100
120
140
160
Time (h) 20
4.0
(b)
3.5
Solvents Production (g/L)
203
C. aminovalericum strain could only grow anaerobically under a 100% N2 atmosphere. The growth profile of C. acetobutylicum YM1 under both conditions (aerobic and anaerobic) was associated with a gradual drop of pH from 6 to 4.66 (under aerobic condition) and 4.70 (under anaerobic condition) in the first 24 h due to the production of organic acids (butyric and acetic acids) during the log phase. Under both conditions, when the growth reached the stationary phase and the metabolism shifted to the solventogenic phase, the pH was increased slightly as a result of the acid reassimilation [25]. The growth profiles under both conditions were similar, and no significant difference was observed (Fig. 2(a)). Moreover, the specific growth rate of C. acetobutylicum YM1 in the first 12 h under aerobic and anaerobic conditions were 0.081 h 1 and 0.089 h 1, respectively. The butyric acid production under both aerobic and anaerobic conditions reached the maximum at 14 h as 3.46 g/L and 4.79 g/L, respectively. The production of acetone, butanol, and ethanol under aerobic conditions were started slightly late, but the final concentrations were similar to that produced under the anaerobic conditions (Fig. 2(b) and (c)). Glucose utilization was increased under anaerobic conditions compared with the aerobic conditions. However, the glucose was completely consumed in the first 48 h under both conditions. The glucose consumption rate under the aerobic conditions at 24 h was 0.84 g/L h and 0.97 g/L h under the anaerobic conditions. The maximum biobutanol production, under both aerobic and anaerobic conditions, was obtained at 120 h as 12.18 g/L and
Acids Production (g/L)
199
15
3.0
2.5 10 2.0
1.5 5
Acids Production (g/L)
198
Acetone Butanol Ethanol ABE Acetic acid Butyric acid
1.0
0.5
0 0
20
40
60
80
100
120
140
160
Time (h) Fig. 1. Profile of ABE fermentation under aerobic conditions (100% initial DO) and anaerobic conditions (0% DO) in a batch culture of C. acetobutylicum YM1: (a) ABE and acid production under aerobic conditions and (b) ABE and acid production under anaerobic conditions.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 4
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
3.5
60
(a)
2.5
40
2.0 30 OD(an-aerobic) OD(aerobic) Glucose (an-aerobic) Glucose (aerobic)
1.5 1.0
20 10
0.5 0.0 0
20
40
60
80
100
120
140
0 160
Time (h) 14
(b)
12
Butanol (g/L)
10 8 Aerobic An-aerobic
6 4 2 0 0
20
40
60
80
100
120
140
160
180
Time (h) 20
(c)
18 16 14
ABE (g/L)
265
The ability of strain YM1 to grow under continuous aeration by air (DO 100%) was investigated in a 5 L bioreactor with a working volume of 3 L. The medium was aerated overnight by air before inoculation, and the DO was maintained at a saturation level of 100% until 12 h after inoculation. No growth or change in pH, acids or solvents production was observed under continuous aeration. When the aeration was stopped and the medium was flushed with nitrogen for 2 min, the culture could revive the growth, acids and solvents production. After the generation of anaerobic conditions, the lag phase took approximately 12 h and was followed by the log phase, which indicated that this strain is aerotolerant, not aerobic. The highest growth was obtained at 48 h with a 2.54 OD600. The maximum final concentrations of biobutanol and ABE were obtained at 96 h from the inoculation (72 h after the growth started) as 10.25 and 16.38 g/L, respectively. The glucose concentration was constant in the first 12 h (under continuous aeration), and after the condition changed to anaerobic, the culture started to consume glucose even though no growth was observed in the first 10 h of glucose consumption. The glucose utilization rate after 24 h was high, and glucose reached 0 g/L in 72 h of culture cultivation (Fig. 3). In another experiment, the YM1 culture was started with anaerobic conditions (0% DO). After 8 h of the cultivation, the culture was sparged with air, and the DO was maintained at 100% DO saturation for 30 min. The data showed that exposure of the culture to the oxygen did not affect the growth or acids production (indicated by pH), and the growth was continued with a similar growth rate under anaerobic conditions (Fig. 4). O’Brien and Morries [16] found that aeration of C. acetobutylicum during the log phase for a period halted the growth and acids production, but the culture could regrow and produce acids after anaerobic conditions were re-established. This result is not in agreement with our study, in which strain YM1 continued to grow and produce acids when exposed to air after 6 h of growth. This study showed that strain YM1 was not affected by aeration after the growth was initiated or initial aerobic conditions (initial DO 100%), but our strain cannot grow under fully continuous aeration. Thus, our strain YM1 is aerotolerant but not fully anaerobic.
267
3.3. Profile of dissolved oxygen during the aerobic fermentation in a 5 L bioreactor
305
The data in this study showed that C. acetobutylicum YM1 is capable of growing and producing biobutanol efficiently with a high initial dissolved oxygen saturation. To study the profile of DO consumption by C. acetobutylicum YM1, a 5 L bioreactor supplemented with a dissolved oxygen meter (Mettler-Toledo, Swiss) was aerated before inoculation until the DO reached 100%. The bioreactor was inoculated by a fresh inoculum of C. acetobutylicum YM1 strain. Immediately after the inoculation, the DO in the medium was dropped gradually with time from 100% DO to 2% DO over 90 min (Fig. 5). The results showed rapid consumption of DO during the growth of strain YM1, and the growth reached 0.913 OD at 600 nm. The increase in growth was 0.067 OD during the 90 min while the decrease in pH was 0.01 at the same time. This mean that even under the presence of oxygen (100% DO saturation), the bacteria was still in the log phase; the bacteria could grow and produce acids as indicated by the drop in pH. Comparing to the growth under anaerobic conditions, the increase in growth and decrease in pH in the first 90 min were similar with a 0.07 increase in OD and 0.1 decrease in pH, respectively. The comparison indicated that the presence of high DO saturation
307
266
50
Glucose (g/L)
Growth (OD 600nm)
3.0
3.2. Effect of continuous aeration on the growth of YM1 and the solvents production
12 10 8
Aerobic An-aerobic
6 4 2
268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
0 0
20
40
60
80
100
120
140
160
180
Time (h) Fig. 2. Profiles of ABE fermentation in a batch culture of YM1 strain under aerobic and anaerobic conditions: (a) growth and glucose consumption, (b) profile of biobutanol production and (c) total ABE production profile.
249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264
12.30 g/L, respectively. The maximum ABE produced from aerobic fermentation of YM1 was 18.09 g/L and was comparable to that produced from anaerobic fermentation (18.49 g/L). The production rate of biobutanol was very high with close values during the first 51 h under aerobic and anaerobic conditions as 0.24 and 0.24 g/L h, respectively; then, the biobutanol generation slowed (Fig. 2(b)). Similarly, ABE production was also high in terms of productivity in the first 51 h, and similar values were obtained under aerobic and anaerobic conditions as 0.36 g/L h and 0.36 g/L h, respectively (Fig. 2(c)). The experimental data of this study showed that the kinetics of growth and solvent production by strain YM1 were similar under anaerobic conditions, which were established by sparging the medium with nitrogen gas until the DO reached 0%, and the initial aerobic condition, which was established by sparging the medium with sterilized air until the DO reached 100%.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
306
308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 5
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
Fig. 3. Effect of continuous aeration on the growth of C. acetobutylicum YM1 and ABE production.
3.0 Aerobic
120
5.5
100 Desolved Oxygen (D.O)
5.0
1.5
4.5 Growth
1.0
80
4.0
pH
D.O (%)
2.0
pH
Growth (OD600 nm)
2.5
6.0
60
40
3.5
0.5
20
3.0
0.0 0
10
20
30
40
50
60
Time (h) Fig. 4. Effect of aerobic conditions at 8 h for 30 min on the growth of C. acetobutylicum YM1.
0 0
20
40
60
80
100
120
Time (min) Fig. 5. The profile of DO in the medium of the YM1 culture during the aerobic fermentation in a 5 L bioreactor.
327 328
329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347
in the medium did not affect the log phase, growth or acid production of the YM1 culture. 3.4. Effect of headspace on biobutanol production under aerobic conditions In this study, the effect of headspace on the growth of C. acetobutylicum YM1 and production of solvent was studied under aerobic conditions (no nitrogen was sparged). The experiments were conducted in 100 mL serum bottles, and the headspace ranges studied were 0%, 25%, 50%, 75%. The inoculum size of all the experiments was 10% (v/v), and the bottles were incubated at 30 °C for 96 h without shaking. Fig. 6(a) shows the effect of headspace on growth and solvent production. The results show that an increase in the headspace led to enhanced growth; the highest growth was found when the headspace was 75%. This can be attributed to the gas pressure in the reactor due to the production of hydrogen and CO2 in the acidogenic phase. A larger headspace allows for a decrease in the pressure of the gas and the stress on the bacterial cells. A similar observation was observed with the production of ABE. There is a correlation between ABE production and the headspace of the reactor; the biggest headspace produced the highest ABE and biobutanol of 15.75 and 10.56 g/L, respectively.
Head space also affects the biobutanol to acetone ratio, and the results showed that an increase of headspace results in a decrease of the biobutanol to acetone ratio (Fig. 6(b)). At a headspace of 0%, the B: A ratio was 3.44 while at a headspace of 75%, the B: A ratio was 2.47. The effect of gas pressure on ABE fermentation was reported by Woods and Jones [26]. It was stated that ABE fermentation under a pressure of 2000 kPa resulted in an increase in the yield of biobutanol while the butyrate yield was decreased. The yield of biobutanol and ethanol but not acetone was reported to be enhanced by increasing the headspace pressure from 100 to 250 kPa [26]. It is clear that the headspace pressure has a significant effect and increasing the headspace pressure in the reactor vessel led to enhanced biobutanol production, which can be attributed to the increase of dissolved hydrogen in the fermentation medium, thus affecting the production of ABE [27]. Under high pressure, C. acetobutylicum produces less primary metabolites (H2 and acids) and increases the solvent as secondary metabolites. Headspace pressure in the fermentation vessel as a stress factor can change the bacterial metabolites and affect the ratio of ABE, as well as force the bacteria to shift from acidogenic phase to solventogenic phase.
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
Growth (OD600) & Solvent Producon (g/L)
6
12
(a)
10
Growth OD Acetone Butanol Ethanol
8 6 4 2 0 0
25
50
75
Head space (%) 4
(b)
B:A rao
3
2
1
saturated by oxygen). It was found that C. acetobutylicum YM1 could grow and produce biobutanol under different DO concentrations. The results showed that increasing the DO in the medium resulted in a decrease of biobutanol production. The data showed that this strain can tolerate the presence of oxygen (DO) in the medium, and it was able to grow and produce ABE even under an initial DO of 100%. As illustrated in Table 1, the C. acetobutylicum YM1 strain growth was similar, and no significant differences in growth were seen even under high DO percentages. The results showed an inverse correlation between DO and biobutanol production (Table 1).The biobutanol production under anaerobic conditions (0% DO) was 11.18 g/L while biobutanol production under 100% DO was 7.89 g/L. In terms of biobutanol yield, the highest biobutanol yield of 0.224 was obtained from 0% DO; a DO of 10% and 25% showed the same yield of 0.221. Increasing the initial DO in the medium resulted in a decrease in the biobutanol yield (Table 1). The biobutanol yield was inversely proportional to DO concentration. Importantly, in a previous study by O’Brien and Morries [16], it was stated that the growth of C. acetobutylicum was ceased under full aerobic conditions. The growth also ceased during a short aeration period in the log phase, but the growth recovered after stopping the aeration with no cell damage being observed. The ability of YM1 (as any solvent-producing Clostridium) to produce gases (H2 and CO2) in the acidogenic phase can contribute to a reduction in the oxygen percentage in the culture during the growth, which trends the conditions to more anaerobic over time.
385
3.6. Effect of agitation speed on biobutanol production
412
The agitation speed under anaerobic conditions was reported to increase the dissolved hydrogen in the medium [27], while in this study agitation speed under aerobic conditions was assumed to increase the dissolved oxygen in the fermentation medium and subsequently facilitate more aerobic conditions. Hence to show the capability of the YM1 strain to grow and produce biobutanol under more aerobiosis, experiments were conducted under an initial DO of 75% in 100 mL serum bottles with a working volume of 50 mL (50% head space), and the culture was agitated after the inoculation at different agitation speeds as shown in Table 2. The results showed that no significance differences in solvents and biobutanol productions were observed when the agitation
413
386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411
0 0
25
50
75
Head space (%) Fig. 6. Effect of headspace on the growth and ABE production under aerobic conditions by C. acetobutylicum YM1: (a) growth and ABE profile and (b) biobutanol to acetone (B:A) ratio.
370 371 372 373 374 375 376
It was found that during ABE fermentation, pressures of up to 105 kPa can be generated naturally due to the elevation of gas formation during the fermentation. Maintenance of positive head space pressure during the fermentation at 105 kPa led to enhanced biobutanol productivity, and the enhancement in biobutanol production was 60% greater compared to non-pressurized cultures [28].
377
3.5. Effect of dissolved oxygen on biobutanol production
378
To study the effect of dissolved oxygen on the growth of C. acetobutylicum YM1 and biobutanol production, various dissolved oxygen percentages were applied to the fermentation medium in sealed serum bottles before autoclaving by sparging filtered air through an air filter (0.2 l pore size) or nitrogen gas (95% N2 and 5% O2) to obtain the desired dissolved oxygen. The DO concentrations were varied from 0% (anaerobic) to 100% (fully
379 380 381 382 383 384
Table 2 Effect of agitation speed on the growth of YM1 and solvent production under aerobic conditions. Agitation speed (rpm)
Growth (OD 600 nm)
Solvent production (g/L) Acetone
Butanol
Ethanol
ABE
0 25 50 100 200
2.196 2.297 2.214 2.218 1.983
3.20 3.91 4.15 4.17 2.31
9.12 9.72 9.12 9.15 7.12
0.90 1.16 0.87 0.97 0.54
13.22 14.79 14.14 14.29 9.97
Table 1 The effect of DO on metabolites production and growth of C. acetobutylicum YM1. DO (%)
0 10 25 50 75 100
Solvent production (g/L)
Acid production (g/L)
Acetone
Butanol
Ethanol
ABE
Acetic acid
Butyric acid
4.68 3.77 3.61 3.17 3.54 3.44
11.18 10.83 10.37 8.16 8.70 7.89
1.15 1.23 1.21 0.71 0.82 0.64
17.00 15.83 15.19 12.03 13.05 11.97
5.17 4.22 4.14 4.80 3.59 4.86
1.25 1.96 1.39 2.44 1.47 1.37
Growth (OD600)
Residual glucose (g/L)
Butanol yield (g/g)
2.36 2.33 2.32 2.36 2.29 2.31
0.00 1.03 3.12 9.21 9.70 7.79
0.224 0.221 0.221 0.200 0.216 0.187
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
414 415 416 417 418 419 420 421 422 423 424
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 7
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440
441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475
speed was between 0 and 100 rpm (Table 2). When the speed was more than 100 rpm, the growth, ABE and biobutanol were decreased compared to agitations of 100 rpm and below. The bacterial cells accumulated in the bottom of the bottle when the agitation speed exceeded 100 rpm. It was reported that agitation of the culture in ABE production is not required due to the sufficient mixing, which results from the gas evolution during the fermentation [28]. This finding supported our results in which no differences were found, in terms of fermentation products, due to varying the agitation. However, our aim was to investigate the effect of agitation as an aeration method. Previous studies have shown that under anaerobic conditions of C. acetobutylicum, high levels of hydrogen supersaturation can be achieved without agitation, and the supersaturation of hydrogen leads to the production of alcohols as reduction products, such as ethanol and biobutanol [29,30]. 3.7. Improved biobutanol production by supplementing the medium with reducing agents under aerobic conditions Chemical reducing agents were added at very low concentrations to support growth and to reach the same level of biobutanol concentrations as obtained from anaerobic fermentation, as well as to avoid the need for nitrogen gas sparging as an economic consideration. To investigate the effect of reducing chemical agents supplementation on ABE fermentation under aerobic conditions, various reducing agents, including resazurine, Na2S, DTT, DTE and potassium ferricyanide, were investigated in a series of experiments with different concentrations (data not shown). Table 3 illustrates the optimized concentrations of each reducing agent, production of ABE and acids, as well as the biobutanol yield. In the control culture of YM1, which was conducted under aerobic conditions without any supplementation, the culture produced 13.89 g/L ABE and 9.96 g/L biobutanol, and the biobutanol yield was 0.199 g/g. Meanwhile, batch fermentation of the YM1 culture under anaerobic conditions resulted in production of 14.62 g/L ABE and 10.61 g/L biobutanol with a biobutanol yield of 0.21 g/L. The increase in biobutanol production under anaerobic conditions was 6.52% compared to the aerobic conditions. Table 3 shows that all of the reducing agents used in this study improved the production of biobutanol and ABE, and the amount produced was higher than that produced under anaerobic conditions. When the culture was supplemented with 0.01 g/L potassium ferricyanide, the production of ABE and biobutanol was 17.54 and 12.94 g/L, respectively, with a biobutanol yield of 0.259 g/g. The yield of biobutanol was increased from 0.199 in the control culture to 0.259 with the addition of 0.01 g/L potassium ferricyanide (30.15%). Potassium ferricyanide is usually used as a physiological buffer to increase the solution’s redox potential, maintain the oxidation–reduction potential and create microaerobic
conditions. The addition of potassium ferricyanide resulted in a cultural conditions less sensitive to oxygen. This is in agreement with that reported by Walden and Hentges [31] who employed C. perfringens. The addition of 0.001 g/L resazurine to the medium resulted in production of 11.17 g/L biobutanol and 15.31 g/L ABE with a biobutanol yield of 0.223 g/g. However, the cell growth was decreased compared to the control culture, which can be attributed to the toxic effect of resazurine on the growth of YM1. DTT and DTE also proved to be good supplementary agents that could enhance the production of biobutanol to 11.60 g/L and 12.40 g/L, as well as improve the growth of YM1 under aerobic conditions (Table 3). Supplementation of the medium with 0.05 g/L Na2S contributed to an increase in ABE and biobutanol production by YM1 under aerobic conditions, and the production of ABE and biobutanol were 17.11 and 12.19 g/L, respectively. The yield of biobutanol was 0.224 g/g, and the cell growth was enhanced 15% compared to the control culture. Hence, it was found that the addition of reducing agents to the fermentation medium under aerobic conditions scavenged the DO and subsequently, improved the biobutanol production to levels higher than that produced under the anaerobic conditions. The results indicate that supplementation with reducing chemical agents at very low concentrations improved the biobutanol and ABE production even under aerobic conditions, and this information can be used to reduce the cost of sparging nitrogen during the ABE fermentation to facilitate anaerobic conditions.
476
3.8. NADH /NADPH oxidase, catalase and superoxide dismutase (SOD) enzyme activity
503
Solvent-producing Clostridium species were reported as non-catalase producers, which is an important enzyme for scavenging the hydrogen peroxide produced via the O2-reduction system [24,32]. Under aerobic conditions, the bacterial growth response to O2 is related to the capability of strains to induce superoxide dismutase (SOD) and NADH/NADPH oxidase enzymes [33]. NADH oxidase and NADPH oxidase activates were investigated as scavengers for hydrogen peroxide in cell-free extracts of the YM1 strain. The results revealed that NADH oxidase and NADPH oxidase showed activities. The activity of NADH oxidase was 49.64 U/mg protein, and the activity of NADPH oxidase was 19.95 U/mg protein (Table 4). These results showed that the NADH/NADPH oxidation system is involved in the O2-reduction system in C. acetobutylicum YM1. Strain YM1 also showed activity of SOD in cell crude extracts, and the SOD activity was 59.02% (% inhibition). Moreover, catalase enzyme was detected in the cell crude extract of this strain, and the catalase activity was 61.19 U/mg protein (Table 4). Brioukhanov et al. reported that most of the investigated Clostridium species and acetogens were negative for catalase enzymes [34]. In contrast, Gaenko et al. reported that some clostridia had catalase enzyme
505
Table 3 Impact of reducing agents in the fermentation of ABE by the YM1 strain under aerobic conditions in flask scale. Reducing agent
Resazurine Na2S DTT DTE Potassium ferricyanide Control (aerobic) Control (anaerobic)
Concentration (g/L)
0.001 0.05 0.3 0.3 0.01
Growth (OD600 nm)
1.87 2.53 2.252 2.207 2.360 2.200 2.251
Solvents (g/L)
Acids (g/L)
Butanol yield (YP/S)
Acetone
Butanol
Ethanol
ABE
Acetic
Butyric
3.11 3.79 3.94 4.24 3.51 3.02 2.92
11.17 12.19 11.60 12.40 12.94 9.96 10.61
1.02 1.12 1.13 1.11 1.09 0.91 1.09
15.31 17.11 16.67 17.75 17.54 13.89 14.62
0.86 0.59 1.12 1.24 1.54 1.01 0.87
0.53 0.57 1.51 1.27 1.32 1.07 0.71
0.223 0.244 0.232 0.248 0.259 0.199 0.210
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502
504
506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 8
N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx
Table 4 Oxygen scavenging enzyme activity in a cell-free extract of YM1 grown under initial 50% DO. Enzyme
Activity
SOD activity (%) Catalase (U/mg Protein) NADH Oxidase (U/mg Protein) NADPH Oxidase (U/mg Protein)
59.02 61.19 49.64 19.95
573
activity [35]. Our strain YM1 exhibited the activity of a catalase enzyme, which is in agreement with Gaenko et al. [35] and not in agreement with Brioukhanov et al. [34]. The data in this study indicated that this strain possesses some enzymes, such as catalase, SOD, NADH oxidase and NADPH oxidase, which are important to protect the bacterial cells against the toxic effect of activated species of oxygen, such as superoxide anion and hydrogen peroxide. These enzymes may be the reason why this strain can tolerate the presence of oxygen. The enzymes responsible for scavenging O2 in anaerobic bacteria typically are NADH oxidase and NADPH oxidase. These enzymes can scavenge O2 to produce water or hydrogen peroxide from a dioxygen reaction [24,36]. C. acetobutylicum, which is an obligatory anaerobic, gram-positive acetone–butanol–ethanol producer, can tolerate air exposure due to its ability to activate its oxidative stress response leading to subsequent rapid cellular O2 consumption and high NADH/NADPH oxidase activity in crude extracts [37]. It was found that the increase in NADH oxidase activity was due to the exposure of C. acetobutylicum to oxygen and was not due to the rise in the culture’s redox potential (Eh) [16]. Aerobic and facultative bacteria have highly regulated antioxidative enzymes, such as catalase and superoxide dismutase. These enzymes decrease the oxygen toxicity and mutagenic effects to maintain the cellular concentration of oxidative metabolites at low levels. Strictly anaerobic bacteria lack the antioxidative enzymes and thus are incapable of surviving oxidative stress [38]. Exposure of C. acetobutylicum to oxygen for short times after the bacteria reached the exponential phase resulted in halted growth, decreased glucose utilization, and inhibited the synthesis of protein, DNA and RNA [16]. At oxygen concentrations of 40 lM, the growth of C. acetobutylicum was inhibited even in a medium composed with dithiothreitol at 50 mV [16]. In the presence of oxygen, only the SOD possessing microorganisms can survive while the catalase enzyme is not so important [39]. Previous studies also revealed that the microorganisms tolerance to oxygen is related to SOD activity, and the higher aerotolerant microorganisms showed higher SOD activity than microorganisms with low SOD activity or microorganisms lacking SOD [40]. In black-pigmented Bacteroides, superoxide dismutase activity exhibited a higher correlation with oxygen tolerance than with the activity of NADH oxidase [41]. However, microorganisms can obviously contain significant superoxide dismutase activity and still be incapable of growth in air. Hence, the presence of superoxide dismutase enzyme is not restricted to microorganisms capable of growth in air [39].
574
4. Conclusion
575
The ability of C. acetobutylicum YM1 strain to grow and produce high concentrations of biobutanol efficiently under conditions that are not fully anaerobic deserves interest, as it has been stated by previous studies that solvent-producing Clostridium strains are obligatory anaerobic. This strain possesses an oxygen scavenging system that is represented by SOD, catalase and NADH/NADPH
527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572
576 577 578 579 580
oxidases. The growth profile and solvent production were similar when the culture was cultivated under aerobic and anaerobic conditions. The addition of small concentrations of reducing agents resulted in a considerable improvement in biobutanol production, as well as a substitute for using anaerobic conditions.
581
Acknowledgment
586
The authors would like to thank University Kebangsaan Malaysia for funding this study under grants; DLP-2013-023, GUP-2013-037, UKM-DIP-2012-30 and UKM-DLP-2012-007.
587
References
590
[1] Kheyrandish M, Asadollahi MA, Jeihanipour A, Doostmohammadi M, RismaniYazdi H, Karimi K. Direct production of acetone–butanol–ethanol from waste starch by free and immobilized Clostridium acetobutylicum. Fuel 2015;142:129–33. [2] Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS. Fermentative butanol production by clostridia. Biotechnol Bioeng 2008;101:209–28. [3] Khanna S, Goyal A, Moholkar VS. Production of n-butanol from biodiesel derived crude glycerol using Clostridium pasteurianum immobilized on Amberlite. Fuel 2013;112:557–61. [4] Al-Shorgani NKN, Ali E, Kalil MS, Yusoff WMW. Bioconversion of butyric acid to butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a limited nutrient medium. Bioenergy Res 2012;5:287–93. [5] Al-Shorgani NKN, Kalil MS, Yusoff WMW. The effect of different carbon sources on biobutanol production using Clostridium saccharoperbutylacetonicum N14. Biotechnology 2011;10:280–5. [6] Chen B-Y, Chuang F-Y, Lin C-L, Chang J-S. Deciphering butanol inhibition to Clostridial species in acclimatized sludge for improving biobutanol production. Biochem Eng J 2012;69:100–5. [7] Lee SY, Lee DY, Kim TY. Systems biotechnology for strain improvement. Trends Biotechnol 2005;23:349–58. [8] Lee S, Park J, Jang S, Nielsen L, Kim J, Jung K. Fermentative butanol production by clostridia. Biotechnol Bioeng 2008;101:209–28. [9] Qureshi N, Blaschek HP. Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation. Appl Biochem Biotechnol 2000;84–86:225–35. [10] Andreesen J, Bahl H, Gottschalk G. Introduction to the physiology and biochemistry of the genus Clostridium. In: Minton N, Clarke D, editors. Clostridia, vol. 3. US: Springer; 1989. p. 27–62. [11] Jones DT, Woods DR. Acetone-butanol fermentation revisited. Microbiol Rev 1986;50:484–524. [12] Kawasaki S, Nakagawa T, Nishiyama Y, Benno Y, Uchimura T, Komagata K, et al. Effect of oxygen on the growth of Clostridium butyricum (type species of the genus Clostridium), and the distribution of enzymes for oxygen and for active oxygen species in Clostridia. J Ferment Bioeng 1998;86:368–72. [13] Yazdani SS, Gonzalez R. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 2007;18:213–9. [14] Bramono SE, Lam YS, Ong SL, He J. A mesophilic Clostridium species that produces butanol from monosaccharides and hydrogen from polysaccharides. Bioresour Technol 2011;102:9558–63. [15] Chua TK, Liang D-W, Qi C, Yang K-L, He J. Characterization of a butanol– acetone-producing Clostridium strain and identification of its solventogenic genes. Bioresour Technol 2013;135:372–8. [16] O’Brien RW, Morris JG. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol 1971;68:307–18. [17] Qureshi N, Blaschek HP. ABE production from corn: a recent economic evaluation. J Ind Microbiol Biotechnol 2001;27:292–7. [18] Qureshi N, Saha BC, Cotta MA, Singh V. An economic evaluation of biological conversion of wheat straw to butanol: a biofuel. Energy Convers Manage 2013;65:456–62. [19] Abdeshahian P, Al-Shorgani NKN, Salih NKM, Shukor H, Kadier A, Hamid AA, et al. The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process. Int J Hydrogen Energy 2014;39:12524–31. [20] Al-Shorgani NKN, Hamid AA, Yusoff WMW, Kalil MS. Pre-optimization of medium for biobutanol production by a new isolate of solvent-producing Clostridium. BioResources 2013;8:1420–30. [21] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [22] Salleh M, Tsuey L, Ariff A. The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 utilizing different pH control strategies. Biotechnol Bioprocess Eng 2008;13:33–9. [23] Kawasaki S, Watamura Y, Ono M, Watanabe T, Takeda K, Niimura Y. Adaptive responses to oxygen stress in obligatory anaerobes Clostridium acetobutylicum and Clostridium aminovalericum. Appl Environ Microbiol 2005;71:8442–50. [24] Kawasaki S, Ishikura J, Chiba D, Nishino T, Niimura Y. Purification and characterization of an H2O-forming NADH oxidase from Clostridium
591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073
582 583 584 585
588 589
JFUE 9303
No. of Pages 9, Model 5G
6 June 2015 N.K.N. Al-Shorgani et al. / Fuel xxx (2015) xxx–xxx 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682
[25]
[26] [27] [28] [29]
[30]
[31]
[32]
aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria. Arch Microbiol 2004;181:324–30. Kumar M, Gayen K, Saini S. Role of extracellular cues to trigger the metabolic phase shifting from acidogenesis to solventogenesis in Clostridium acetobutylicum. Bioresour Technol 2013;138:55–62. Woods DR, Jones DT. Physiological responses of Bacteroides and Clostridium strains to environmental stress factors. Adv Microb Physiol 1986;28:1–64. Doremus MG, Linden JC, Moreira AR. Agitation and pressure effects on acetone–butanol fermentation. Biotechnol Bioeng 1985;27:852–60. Maddox IS. The acetone–butanol–ethanol fermentation: recent progress in technology. Biotechnol Genet Eng Rev 1989;7:189–220. Lamed R, Lobos J, Su T. Effects of stirring and hydrogen on fermentation products of Clostridium thermocellum. Appl Environ Microbiol 1988;54:1216–21. Lobos J, Lamed RJ, Su TM. H2 supersaturation: its effect on end product formation in anaerobic fermentations. Annual Meeting of the American Society for Microbiology. Atlanta; 1982. p. 198. Walden WC, Hentges DJ. Differential effects of oxygen and oxidation reduction potential on the multiplication of three species of anaerobic intestinal bacteria. Appl Microbiol 1975;30:781–5. Keis S, Shaheen R, Jones DT. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov. Int J Syst Evol Microbiol 2001;51:2095–103.
9
[33] Higuchi M, Shimada M, Yamamoto Y, Hayashi T, Koga T, Kamio Y. Identification of two distinct NADH oxidases corresponding to H2O2-forming oxidase and H2O-forming oxidase induced in Streptococcus mutans. J Gen Microbiol 1993;139:2343–51. [34] Brioukhanov AL, Thauer RK, Netrusov AI. Catalase and superoxide dismutase in the cells of strictly anaerobic microorganisms. Microbiology 2002;71:281–5. [35] Gaenko GP, Reshetnikova IV, Duda VI, Plekhanova IO, Gusev MV. Superoxide dismutase in Clostridium butyricum spores. Mikrobiologiia 1985;54:322–4. [36] Reed DW, Millstein J, Hartzell PL. H2O2-forming NADH oxidase with diaphorase (cytochrome) activity from Archaeoglobus fulgidus. J Bacteriol 2001;183:7007–16. [37] Hillmann F, Fischer R-J, Saint-Prix F, Girbal L, Bahl H. PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum. Mol Microbiol 2008;68:848–60. [38] Brioukhanov AL, Netrusov AI. Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review. Appl Biochem Microbiol 2007;43:567–82. [39] Hewitt J, Morris JG. Superoxide dismutase in some obligately anaerobic bacteria. FEBS Lett 1975;50:315–8. [40] Privalle CT, Gregory EM. Superoxide dismutase and O2 lethality in Bacteroides fragilis. J Bacteriol 1979;138:139–45. [41] Amano A, Tamagawa H, Takagaki M, Murakami Y, Shizukuishi S, Tsunemitsu A. Relationship between enzyme activities involved in oxygen metabolism and oxygen tolerance in black-pigmented bacteroides. J Dent Res 1988;67:1196–9.
683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707
Please cite this article in press as: Al-Shorgani NKN et al. Biobutanol production by a new aerotolerant strain of Clostridium acetobutylicum YM1 under aerobic conditions. Fuel (2015), http://dx.doi.org/10.1016/j.fuel.2015.05.073