- Email: [email protected]
Using Glycerol as a Sole Carbon Source for Clostridium beijerinckii Fermentation
Available online at www.sciencedirect.com
ScienceDirect
Availableonline onlineatatwww.sciencedirect.com www.sciencedirect.com Available Energy Procedia 00 (2017) 000–000
ScienceDirect ScienceDirect
www.elsevier.com/locate/procedia
Energy Procedia 138 Energy Procedia 00(2017) (2017)1105–1109 000–000 www.elsevier.com/locate/procedia
2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies 2017 AEDCEE, 25 - 26 May 2017, Bangkok, Thailand
Glycerol as a Sole Carbon Source TheUsing 15th International Symposium on District Heating and Cooling
for Clostridium beijerinckii Fermentation Assessing the feasibility of using the heat demand-outdoor a, b Sanguanchaipaiwong *, district Noppol Leksawasdi temperatureVorapat function for a long-term heat demand forecast Department of Biology, Faculty of science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand a,b,c a a b c c Bioprocess Research Cluster, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50100, Thailand aa
I. Andrić
bbBioprocess a
*, A. Pina , P. Ferrão , J. Fournier ., B. Lacarrière , O. Le Corre
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal b Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France c Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France
Abstract
Butanol is a promising biofuel with its fuel properties which are similar to gasoline and able to be synthesized from acetonebutanol-ethanol fermentation of Clostridium sp. This study has been focused on using glycerol, a by-product from biodiesel manufacturing as a carbon source for Clostridium beijerinckii TISTR 1390. The culture was cultivated at 37°C in P2 medium with Abstract 20-60 g/L glycerol under anaerobic condition, compared with glucose as a control. Samples were collected periodically for the analysis of viable cell concentration, reducing sugar and metabolite concentrations. It has been found that C. beijerinckii grown District heating networks are 168 commonly addressed in the literature of thehas most effective decreasing of the much slower in glycerol. After h cultivation, the cell growth in 20 as g/Lone glycerol achieved the solutions maximumfor concentration In P2 medium glucose, the systems amount of viablehigh cellinvestments (3.93 x 1066 which CFU/mL 20 g/L glucose) 4.78 × 1066 CFU/mL. greenhouse gas emissions from thecontaining building sector. These require arewith returned through the had heat sales. Due to the changed conditions and period building renovation heat be demand in the could decrease, reached the maximum level atclimate 24 h. The much longer for highest cell policies, number might the reason thatfuture C. beijerinckii could not produce the butanol from merely glycerol. On the other hand, the butanol concentration of 8.12 g/L was obtained from 60 g/L prolonging investment return period. glucose. Glycerol probably induced C. beijerinckii in switching a reductive pathway. The main scope of this paper is to assess the feasibility of usingtothe heat demand – outdoor temperature function for heat demand districtPublished of Alvalade, locatedLtd. in Lisbon (Portugal), was used as a case study. The district is consisted of 665 ©forecast. 2017 TheThe Authors. by Elsevier ©buildings 2017 The Authors. Published by Elsevierperiod Ltd. and typology. Three weather scenarios (low, medium, high) and three district vary in both construction Peer-reviewthat under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in Peer-review under responsibility of the Organizing Committee ofdeep). 2017 AEDCEE. scenarios wereEmerging developed (shallow, intermediate, To estimate the error, obtained heat demand values were Drenovation eveloping Countries and Economies. compared with results from a dynamic heat demand model, previously developed and validated by the authors. Keywords: Glycerol; Clostridium acetone-butanol-ethanol fermentation; anaerobe; carbon source The results showed that whenbeijerinckii; only weather change is considered, the margin of error could be acceptable for some applications (the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered). 1.The Introduction value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the decrease in the number of heating hours of 22-139h during the heating season (depending on the combination of weather and Recently,scenarios glycerolconsidered). has been On produced largefunction quantity as a by-product 10%) (depending from biodiesel renovation the otherinhand, intercept increased for(approximately 7.8-12.7% per decade on the coupled scenarios). The pose valuesasuggested could be used to modify the function abundant parametersglycerol for the scenarios production that might major environmental problem. However, could beconsidered, used as and an improve the accuracy of heat demand estimations. © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and * Corresponding author. Tel.: +0-662-329-8400; fax: +0-662-329-8427. Cooling. E-mail address: [email protected]; [email protected]
Keywords: Heat demand; Forecast; Climate change 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling. 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies. 10.1016/j.egypro.2017.10.127
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 Sanguanchaiapiwong V. and Leksawasdi N./ Energy Procedia 00 (2017) 000–000
1106 2
economical carbon source for fermentation. Glycerol has usually been utilized as a microbial substrate for the production of industrial chemicals, for example, 1,3-propanediol, succinic acid, propionic acid, citric acid, and ethanol [1]. There is a possibility to utilize glycerol directly for butanol production [2-4]. Global energy crisis and limited supply of fossil fuels have been the main focus for scientific research to sustainable and alternative fuel production. Utilization of renewable substrate would bring great chance for the development of a sustainable and economical biofuel production. Butanol is a potential alternative fuel due to the likeness of its petrol properties to gasoline [5]. It is non-corrosive, has comparable energy with gasoline, and low vapor pressure. Butanol is an essential industrial chemical and has been used as a precursor for many chemicals in various industries [5]. Clostridium sp. could produce butanol via acetone-butanol-ethanol (ABE) fermentation [6-8] from C5 and C6 sugars [9]. The most recognized solventogenic species for butanol production are Clostridium acetobutylicum and C. beijerinckii. This research is one of the few reports [4,10,11] that focused on evaluation of glycerol utilization by C. beijerinckii for growth and solvent production. The effect of glycerol fermentation on cells growth and solvent production had been investigated for the first time for C. beijerinckii TISTR1390. 2. Materials and methods 2.1. Bacteria and Medium Clostridium beijerinckii TISTR 1390 had been kindly provided by Culture Collection of Thailand Institute of Scientific and Technological Research (TISTR). It was transferred and conserved in reinforced clostridial medium (RCM, Difco™) at 4°C every 4 weeks and kept in glycerol stock at -70°C. P2 medium [12] was employed for C. beijerinckii growth and ABE production and composed of (per L) 20-60 g glucose, 1 g yeast extract, 0.5 g KH2PO4, 0.5 g K2HPO4, 2.2 g ammonium acetate, 0.2 g MgSO4•7H2O, 0.01 g MnSO4•H2O, 0.01 g FeSO4•7H2O, 0.01 g NaCl, 1 mg para-amino-benzoic acid, 1 mg thiamine, and 10 µg biotin. Vitamins and minerals have been sterilized by membrane filtration. The other components were sterilized at 121°C, 15 psi. Various glycerol concentration levels (20-60 g/L) were added into P2 medium instead of glucose. 2.2. Inoculum Preparation and ABE fermentation Stock culture was subcultured to 5 mL RCM and heat-shocked at 80°C for 10 min. After incubating at 37°C for 48 h, C. beijerinckii was transferred to 45 mL RCM and cultivated at 37°C for 24 h in anaerobic condition and used as an inoculum for ABE fermentation. The culture was inoculated for 10% (v/v) in P2 medium and the fermentation was carried out in 250 mL flask and incubated anaerobically at 37°C for 168 h (glucose) and for 360 h (glycerol). 2.3. Chemical Analyses The samples were collected in triplicate on a daily basis and measured for viable cells count by pour plate technique (in CFU/mL). Subsequently, the culture samples were centrifuged at 3,000 rpm (2,822 x g) for 15 min. The supernatants were analyzed for reducing sugar concentration by 3,5-dinitrosalicylic acid method [13] and the concentration of glycerol and ABE by HPLC with Aminex® HPX-87H Ion Exclusion column (7.8 × 300 mm) with refractive index detector, 37°C. 5 mM H2SO4 as mobile phase at 0.75 mL/min. The injection volume was 20 µL. 2.4. Kinetic Parameters The determined kinetic parameters included specific growth rate ( µ , h-1), productivity (g/L⋅ h), yield coefficients for cell growth ( Yx s , CFU/g), butanol and acetone-butanol (AB) production ( Y p s , g/g) [14] using following equations.
ln(xmax x0 ) t p − p0 Productivity = max t
µ=
(1) (2)
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 V. Sanguanchaiapiwong and N. Leksawasdi/ Energy Procedia 00 (2017) 000–000
1107 3
Yx s =
xmax − x0 s0 − s
(3)
Yp s =
pmax − p0 s0 − s
(4)
Where xmax and x0 were the maximum and initial number of viable cells (CFU/mL), s and s0 represented final and
initial substrate concentration (g/L), pmax and p 0 were the maximum and initial concentration of butanol or AB (g/mL) and t was time of cell growth or butanol or AB production (h). 3. Results and discussions 3.1 The effect of glycerol on C. beijerinckii growth
The profiles of viable cells count based on the anaerobic fermentation of C. beijerinckii TISTR 1390 at 37°C using 20-60 g/L glucose or glycerol as carbon sources have been shown and compared in Fig. 1 and 2. From Fig. 1a and 1b, the growth of C. beijerinckii using glucose was faster than glycerol. After 168 h cultivation, the growth in 20 g/L glycerol reached the maximum cells number of 4.786 × 106 CFU/mL. This was compared to P2 medium containing 20 g/L glucose with viable cells concentration of 3.93 x 106 CFU/mL after 24 h. The decline of growth from the maximum was observed when glucose was used as carbon source (Fig. 1a). Since the inoculum was prepared from RCM containing glucose, the culture in glycerol might need time to adjust its metabolism to accommodate itself which corresponded to the situation where butanol was absent in section 3.2.
Fig. 1. Time profiles of C. beijerinckii TISTR 1390 anaerobically cultivated in P2 medium added with (a) glucose or (b) glycerol at 37°C.
C. beijerinckii NRRL B593 has been reported to grow in crude glycerol from biodiesel factory [11]. The maximum OD620 nm was approximately 1.40 within 24 h from 20 g/L glycerol and the microbe was able to consume glycerol completely. In this study, the concentration of glycerol was not depleted (8.82 g/L) at the end of fermentation. The dependency of C. beijerinckii strains on glycerol consumption was evident. C. beijerinckii ATCC 11941 and ATCC 14949 could be propagated through four serial sub-culturing in 1% glycerol which were compared to the lack of growth for ATCC 858 and ATCC 25752 [10]. The maximum cells concentration of C. beijerinckii TISTR 1390 fermentation in glucose could be compared to glycerol. The maximum cells concentration increased as the glucose concentration increased. On the contrary, once the amount of glycerol rose in the cultivation using glycerol, the maximum level of viable cells decreased. The highest cells number was obtained from the cultivation with 60 g/L glucose at 24 h.
1108 4
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 Sanguanchaiapiwong V. and Leksawasdi N./ Energy Procedia 00 (2017) 000–000
Fig. 2. Cell growth of C. beijerinckii TISTR 1390 using glucose or glycerol as sole carbon source at 37°C and anaerobic condition.
3.2 The effect of glycerol on Acetone-Butanol Production To investigate how glycerol influenced product synthesis of C. beijerinckii TISTR 1390, anaerobic cultivations were carried out at 37°C. Fig. 3 shows the acetone - butanol production during 60 h. P2 medium, which contained 60 g/L glucose, resulted in maximum concentrations of acetone (1.63 g/L) and butanol (8.12 g/L) with C. beijerinckii. Acetone concentration of 3.80 g/L was obtained from 20 g/L glycerol. Ethanol production was not observed for all cultivations of C. beijerinckii. There has been report that C. beijerinckii NRRL B593 scarcely produced ethanol [11]. The main product of that report was 1,3-propanediol (1,3-PD) with the concentration of 10 g/L in 24 h of cultivation. The yield of 1,3-PD to raw glycerol was 0.6 mol/mol and the productivity was 0.5 g/L⋅ h. Glycerol has greatly reduced characteristic. Once utilizing as a carbon source, glycerol could be converted to glycolytic intermediates, phosphoenolpyruvate, or pyruvate, creating twice the quantity of reducing equivalents compared to those generated from sugars [15]. These intermediates could lead to the production of 1,3-PD via reductive pathway [16]. The higher concentration of glycerol might lead to higher 1,3-PD production, rather than oxidative pathway which produced butanol.
Fig. 3. Acetone-butanol production of Clostridium beijerinckii TISTR 1390 using (a) glucose or (b) glycerol at 37°C and anaerobic condition.
From Table 1, the specific growth rate ( µ ) calculated from glucose (0.178 h-1) was much higher than that of glycerol (0.005 h-1) due to shorter cultivation time. Butanol yield of 0.203 g/g glucose and productivity of 0.068 g/L⋅h were obtained. C. pasteurianum ATCC 6013 was able to grow on crude glycerol with the average butanol yield of 0.24 g/g glycerol within 24 days [4]. The relatively low productivity (approximately 0.0075 g/L⋅h) corresponded to the slow growth rate of this strain based on glycerol.
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 V. Sanguanchaiapiwong and N. Leksawasdi/ Energy Procedia 00 (2017) 000–000
1109 5
Table 1. Kinetic parameters of Clostridium beijerinckii TISTR 1390 growth and AB production. Kinetics Parameters
Glucose
µ (h-1)
0.178
Yx s (CFU/g) Y p s of butanol (g/g)
1.60 × 10 0.203
Glycerol 0.005 5
3.70 × 105 0
Butanol productivity (g/L⋅h)
0.068
0
Y p s of AB (g/g) AB productivity (g/L⋅h)
0.243
0.522
0.081
0.023
4. Conclusions Glycerol could be utilized by Clostridium beijerinckii TISTR 1390 to promote cell growth but direct use for butanol production was not evident. Two-steps fermentation might be investigated further by adding sugars to induce butanol production at a later stage after glycerol had been used to promote sufficient cell growth. Acknowledgements The authors would like to acknowledge the financial support or supports from National Research Council of Thailand (NRCT) and Faculty of Science, KMITL (Sanguanchaipaiwong V.) as well as National Research University (NRU) - Chiang Mai University (CMU), NRU - Office of Higher Education Commission (OHEC), Ministry of Education, CMU, Thailand (Leksawasdi N.) for this research. References [1] Jang YS, Malaviya A, Cho C, Lee J, Lee SY. Butanol production from renewable biomass by clostridia. Bioresour Technol 2012;123:653-663. [2] Biebl H. Fermentation of glycerol by Clostridium pasteurianum: batch and continuous culture studies. J Ind Microbiol Biotechnol 2001;27:1826. [3] Dabrock B, Bahl H, Gottschalk G. Parameters affecting solvent production by Clostridium pasteurianum. Appl Environ Microbiol 1992;58:1233-1239. [4] Taconi KA, Venkataramanan KP, Johnson DT. Growth and solvent production by Clostridium parteurianum ATCC® 6013™ utilizing biodiesel-derived crude glycerol as the sole carbon source. Environ Prog Sustain Energy. 2009; 28:100-110. [5] Zhu JH, Yang F. Biological process for butanol production. In: Cheng J, editor. Biomass to renewable energy processes. New York: CRC Press: 2009. p. 271-336. [6] Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS. Fermentative butanol production by Clostridia. Biotechnol Bioeng 2008;101:209– 228. [7] Bellodo C, Infante C, Coca M, González-Benito G, Lucas S, García-Cubero MT. Efficient acetone-butanol-ethanol production by Clostridium beijerinckii from sugar beet pulp. Bioresour Technol 2015;190:332-338. [8] Cheng HH, Whang LM, Chan KC, Chung MC, Wu SH, Liu CP, Tien SY, Chen SY, Chang JS, Lee WJ. Biological butanol production from microalgae-based biodiesel residues by Clostridium acetobutylicum. Bioresour Technol 2015;184:379-385. [9] Lin DS, Yen HW, Kao WC, Cheng CL, Chen WM, Huang CC, Chang JS. Bio-butanol production from glycerol with Clostridium pasteurianum CH4: the effects of butyrate addition and in situ butanol removal via membrane distillation. Biotechnol Biofuels 2015;8:168-179. [10] Forsberg CW. Production of 1,3-propanediol from glycerol by Clostridium acetobutylicum and other Clostridium species. Appl Environ Microbiol 1987;53:639-643. [11] Gungormusler M, Gonen C, Azbar N. 1,3-Propanediol production potential by a locally isolated strain of Klebsiella pneumonia in comparison to Clostridium beijerinckii NRRL B593 from waste glycerol. J Polym Environ 2011;19:812-817. [12] Qureshi N, Blaschek HP. Butanol recovery from model solution/fermentation broth by pervaporation: evaluation of membrane performance. Biomass Bioenergy 1999;17:175–184. [13] Miller GL. Use of dinitrosaIicyIic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–428. [14] Hong J. Yield Coefficients for Cell Mass and Product Formation. Biotechnol Bioeng 1989;33:506–507. [15] Yazdani SS, Gonzalez R. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 2007;18:213-219. [16] Quilaguy-Ayure DM, Montoya-Solano JD, Suárez-Moreno ZR, Bernal-Morales JM, Montoya-Castaño D. Analysing the dhaT gene in Colombian Clostridium sp. (Clostridia) 1,3-propanediol-producing strains. Univ Sci 2010;15:17-26.
ScienceDirect
Availableonline onlineatatwww.sciencedirect.com www.sciencedirect.com Available Energy Procedia 00 (2017) 000–000
ScienceDirect ScienceDirect
www.elsevier.com/locate/procedia
Energy Procedia 138 Energy Procedia 00(2017) (2017)1105–1109 000–000 www.elsevier.com/locate/procedia
2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies 2017 AEDCEE, 25 - 26 May 2017, Bangkok, Thailand
Glycerol as a Sole Carbon Source TheUsing 15th International Symposium on District Heating and Cooling
for Clostridium beijerinckii Fermentation Assessing the feasibility of using the heat demand-outdoor a, b Sanguanchaipaiwong *, district Noppol Leksawasdi temperatureVorapat function for a long-term heat demand forecast Department of Biology, Faculty of science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand a,b,c a a b c c Bioprocess Research Cluster, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50100, Thailand aa
I. Andrić
bbBioprocess a
*, A. Pina , P. Ferrão , J. Fournier ., B. Lacarrière , O. Le Corre
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal b Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France c Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France
Abstract
Butanol is a promising biofuel with its fuel properties which are similar to gasoline and able to be synthesized from acetonebutanol-ethanol fermentation of Clostridium sp. This study has been focused on using glycerol, a by-product from biodiesel manufacturing as a carbon source for Clostridium beijerinckii TISTR 1390. The culture was cultivated at 37°C in P2 medium with Abstract 20-60 g/L glycerol under anaerobic condition, compared with glucose as a control. Samples were collected periodically for the analysis of viable cell concentration, reducing sugar and metabolite concentrations. It has been found that C. beijerinckii grown District heating networks are 168 commonly addressed in the literature of thehas most effective decreasing of the much slower in glycerol. After h cultivation, the cell growth in 20 as g/Lone glycerol achieved the solutions maximumfor concentration In P2 medium glucose, the systems amount of viablehigh cellinvestments (3.93 x 1066 which CFU/mL 20 g/L glucose) 4.78 × 1066 CFU/mL. greenhouse gas emissions from thecontaining building sector. These require arewith returned through the had heat sales. Due to the changed conditions and period building renovation heat be demand in the could decrease, reached the maximum level atclimate 24 h. The much longer for highest cell policies, number might the reason thatfuture C. beijerinckii could not produce the butanol from merely glycerol. On the other hand, the butanol concentration of 8.12 g/L was obtained from 60 g/L prolonging investment return period. glucose. Glycerol probably induced C. beijerinckii in switching a reductive pathway. The main scope of this paper is to assess the feasibility of usingtothe heat demand – outdoor temperature function for heat demand districtPublished of Alvalade, locatedLtd. in Lisbon (Portugal), was used as a case study. The district is consisted of 665 ©forecast. 2017 TheThe Authors. by Elsevier ©buildings 2017 The Authors. Published by Elsevierperiod Ltd. and typology. Three weather scenarios (low, medium, high) and three district vary in both construction Peer-reviewthat under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in Peer-review under responsibility of the Organizing Committee ofdeep). 2017 AEDCEE. scenarios wereEmerging developed (shallow, intermediate, To estimate the error, obtained heat demand values were Drenovation eveloping Countries and Economies. compared with results from a dynamic heat demand model, previously developed and validated by the authors. Keywords: Glycerol; Clostridium acetone-butanol-ethanol fermentation; anaerobe; carbon source The results showed that whenbeijerinckii; only weather change is considered, the margin of error could be acceptable for some applications (the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered). 1.The Introduction value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the decrease in the number of heating hours of 22-139h during the heating season (depending on the combination of weather and Recently,scenarios glycerolconsidered). has been On produced largefunction quantity as a by-product 10%) (depending from biodiesel renovation the otherinhand, intercept increased for(approximately 7.8-12.7% per decade on the coupled scenarios). The pose valuesasuggested could be used to modify the function abundant parametersglycerol for the scenarios production that might major environmental problem. However, could beconsidered, used as and an improve the accuracy of heat demand estimations. © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and * Corresponding author. Tel.: +0-662-329-8400; fax: +0-662-329-8427. Cooling. E-mail address: [email protected]; [email protected]
Keywords: Heat demand; Forecast; Climate change 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling. 1876-6102 © 2017 The Authors. Published by Elsevier Ltd. Peer-review under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in Developing Countries and Emerging Economies. 10.1016/j.egypro.2017.10.127
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 Sanguanchaiapiwong V. and Leksawasdi N./ Energy Procedia 00 (2017) 000–000
1106 2
economical carbon source for fermentation. Glycerol has usually been utilized as a microbial substrate for the production of industrial chemicals, for example, 1,3-propanediol, succinic acid, propionic acid, citric acid, and ethanol [1]. There is a possibility to utilize glycerol directly for butanol production [2-4]. Global energy crisis and limited supply of fossil fuels have been the main focus for scientific research to sustainable and alternative fuel production. Utilization of renewable substrate would bring great chance for the development of a sustainable and economical biofuel production. Butanol is a potential alternative fuel due to the likeness of its petrol properties to gasoline [5]. It is non-corrosive, has comparable energy with gasoline, and low vapor pressure. Butanol is an essential industrial chemical and has been used as a precursor for many chemicals in various industries [5]. Clostridium sp. could produce butanol via acetone-butanol-ethanol (ABE) fermentation [6-8] from C5 and C6 sugars [9]. The most recognized solventogenic species for butanol production are Clostridium acetobutylicum and C. beijerinckii. This research is one of the few reports [4,10,11] that focused on evaluation of glycerol utilization by C. beijerinckii for growth and solvent production. The effect of glycerol fermentation on cells growth and solvent production had been investigated for the first time for C. beijerinckii TISTR1390. 2. Materials and methods 2.1. Bacteria and Medium Clostridium beijerinckii TISTR 1390 had been kindly provided by Culture Collection of Thailand Institute of Scientific and Technological Research (TISTR). It was transferred and conserved in reinforced clostridial medium (RCM, Difco™) at 4°C every 4 weeks and kept in glycerol stock at -70°C. P2 medium [12] was employed for C. beijerinckii growth and ABE production and composed of (per L) 20-60 g glucose, 1 g yeast extract, 0.5 g KH2PO4, 0.5 g K2HPO4, 2.2 g ammonium acetate, 0.2 g MgSO4•7H2O, 0.01 g MnSO4•H2O, 0.01 g FeSO4•7H2O, 0.01 g NaCl, 1 mg para-amino-benzoic acid, 1 mg thiamine, and 10 µg biotin. Vitamins and minerals have been sterilized by membrane filtration. The other components were sterilized at 121°C, 15 psi. Various glycerol concentration levels (20-60 g/L) were added into P2 medium instead of glucose. 2.2. Inoculum Preparation and ABE fermentation Stock culture was subcultured to 5 mL RCM and heat-shocked at 80°C for 10 min. After incubating at 37°C for 48 h, C. beijerinckii was transferred to 45 mL RCM and cultivated at 37°C for 24 h in anaerobic condition and used as an inoculum for ABE fermentation. The culture was inoculated for 10% (v/v) in P2 medium and the fermentation was carried out in 250 mL flask and incubated anaerobically at 37°C for 168 h (glucose) and for 360 h (glycerol). 2.3. Chemical Analyses The samples were collected in triplicate on a daily basis and measured for viable cells count by pour plate technique (in CFU/mL). Subsequently, the culture samples were centrifuged at 3,000 rpm (2,822 x g) for 15 min. The supernatants were analyzed for reducing sugar concentration by 3,5-dinitrosalicylic acid method [13] and the concentration of glycerol and ABE by HPLC with Aminex® HPX-87H Ion Exclusion column (7.8 × 300 mm) with refractive index detector, 37°C. 5 mM H2SO4 as mobile phase at 0.75 mL/min. The injection volume was 20 µL. 2.4. Kinetic Parameters The determined kinetic parameters included specific growth rate ( µ , h-1), productivity (g/L⋅ h), yield coefficients for cell growth ( Yx s , CFU/g), butanol and acetone-butanol (AB) production ( Y p s , g/g) [14] using following equations.
ln(xmax x0 ) t p − p0 Productivity = max t
µ=
(1) (2)
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 V. Sanguanchaiapiwong and N. Leksawasdi/ Energy Procedia 00 (2017) 000–000
1107 3
Yx s =
xmax − x0 s0 − s
(3)
Yp s =
pmax − p0 s0 − s
(4)
Where xmax and x0 were the maximum and initial number of viable cells (CFU/mL), s and s0 represented final and
initial substrate concentration (g/L), pmax and p 0 were the maximum and initial concentration of butanol or AB (g/mL) and t was time of cell growth or butanol or AB production (h). 3. Results and discussions 3.1 The effect of glycerol on C. beijerinckii growth
The profiles of viable cells count based on the anaerobic fermentation of C. beijerinckii TISTR 1390 at 37°C using 20-60 g/L glucose or glycerol as carbon sources have been shown and compared in Fig. 1 and 2. From Fig. 1a and 1b, the growth of C. beijerinckii using glucose was faster than glycerol. After 168 h cultivation, the growth in 20 g/L glycerol reached the maximum cells number of 4.786 × 106 CFU/mL. This was compared to P2 medium containing 20 g/L glucose with viable cells concentration of 3.93 x 106 CFU/mL after 24 h. The decline of growth from the maximum was observed when glucose was used as carbon source (Fig. 1a). Since the inoculum was prepared from RCM containing glucose, the culture in glycerol might need time to adjust its metabolism to accommodate itself which corresponded to the situation where butanol was absent in section 3.2.
Fig. 1. Time profiles of C. beijerinckii TISTR 1390 anaerobically cultivated in P2 medium added with (a) glucose or (b) glycerol at 37°C.
C. beijerinckii NRRL B593 has been reported to grow in crude glycerol from biodiesel factory [11]. The maximum OD620 nm was approximately 1.40 within 24 h from 20 g/L glycerol and the microbe was able to consume glycerol completely. In this study, the concentration of glycerol was not depleted (8.82 g/L) at the end of fermentation. The dependency of C. beijerinckii strains on glycerol consumption was evident. C. beijerinckii ATCC 11941 and ATCC 14949 could be propagated through four serial sub-culturing in 1% glycerol which were compared to the lack of growth for ATCC 858 and ATCC 25752 [10]. The maximum cells concentration of C. beijerinckii TISTR 1390 fermentation in glucose could be compared to glycerol. The maximum cells concentration increased as the glucose concentration increased. On the contrary, once the amount of glycerol rose in the cultivation using glycerol, the maximum level of viable cells decreased. The highest cells number was obtained from the cultivation with 60 g/L glucose at 24 h.
1108 4
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 Sanguanchaiapiwong V. and Leksawasdi N./ Energy Procedia 00 (2017) 000–000
Fig. 2. Cell growth of C. beijerinckii TISTR 1390 using glucose or glycerol as sole carbon source at 37°C and anaerobic condition.
3.2 The effect of glycerol on Acetone-Butanol Production To investigate how glycerol influenced product synthesis of C. beijerinckii TISTR 1390, anaerobic cultivations were carried out at 37°C. Fig. 3 shows the acetone - butanol production during 60 h. P2 medium, which contained 60 g/L glucose, resulted in maximum concentrations of acetone (1.63 g/L) and butanol (8.12 g/L) with C. beijerinckii. Acetone concentration of 3.80 g/L was obtained from 20 g/L glycerol. Ethanol production was not observed for all cultivations of C. beijerinckii. There has been report that C. beijerinckii NRRL B593 scarcely produced ethanol [11]. The main product of that report was 1,3-propanediol (1,3-PD) with the concentration of 10 g/L in 24 h of cultivation. The yield of 1,3-PD to raw glycerol was 0.6 mol/mol and the productivity was 0.5 g/L⋅ h. Glycerol has greatly reduced characteristic. Once utilizing as a carbon source, glycerol could be converted to glycolytic intermediates, phosphoenolpyruvate, or pyruvate, creating twice the quantity of reducing equivalents compared to those generated from sugars [15]. These intermediates could lead to the production of 1,3-PD via reductive pathway [16]. The higher concentration of glycerol might lead to higher 1,3-PD production, rather than oxidative pathway which produced butanol.
Fig. 3. Acetone-butanol production of Clostridium beijerinckii TISTR 1390 using (a) glucose or (b) glycerol at 37°C and anaerobic condition.
From Table 1, the specific growth rate ( µ ) calculated from glucose (0.178 h-1) was much higher than that of glycerol (0.005 h-1) due to shorter cultivation time. Butanol yield of 0.203 g/g glucose and productivity of 0.068 g/L⋅h were obtained. C. pasteurianum ATCC 6013 was able to grow on crude glycerol with the average butanol yield of 0.24 g/g glycerol within 24 days [4]. The relatively low productivity (approximately 0.0075 g/L⋅h) corresponded to the slow growth rate of this strain based on glycerol.
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 138 (2017) 1105–1109 V. Sanguanchaiapiwong and N. Leksawasdi/ Energy Procedia 00 (2017) 000–000
1109 5
Table 1. Kinetic parameters of Clostridium beijerinckii TISTR 1390 growth and AB production. Kinetics Parameters
Glucose
µ (h-1)
0.178
Yx s (CFU/g) Y p s of butanol (g/g)
1.60 × 10 0.203
Glycerol 0.005 5
3.70 × 105 0
Butanol productivity (g/L⋅h)
0.068
0
Y p s of AB (g/g) AB productivity (g/L⋅h)
0.243
0.522
0.081
0.023
4. Conclusions Glycerol could be utilized by Clostridium beijerinckii TISTR 1390 to promote cell growth but direct use for butanol production was not evident. Two-steps fermentation might be investigated further by adding sugars to induce butanol production at a later stage after glycerol had been used to promote sufficient cell growth. Acknowledgements The authors would like to acknowledge the financial support or supports from National Research Council of Thailand (NRCT) and Faculty of Science, KMITL (Sanguanchaipaiwong V.) as well as National Research University (NRU) - Chiang Mai University (CMU), NRU - Office of Higher Education Commission (OHEC), Ministry of Education, CMU, Thailand (Leksawasdi N.) for this research. References [1] Jang YS, Malaviya A, Cho C, Lee J, Lee SY. Butanol production from renewable biomass by clostridia. Bioresour Technol 2012;123:653-663. [2] Biebl H. Fermentation of glycerol by Clostridium pasteurianum: batch and continuous culture studies. J Ind Microbiol Biotechnol 2001;27:1826. [3] Dabrock B, Bahl H, Gottschalk G. Parameters affecting solvent production by Clostridium pasteurianum. Appl Environ Microbiol 1992;58:1233-1239. [4] Taconi KA, Venkataramanan KP, Johnson DT. Growth and solvent production by Clostridium parteurianum ATCC® 6013™ utilizing biodiesel-derived crude glycerol as the sole carbon source. Environ Prog Sustain Energy. 2009; 28:100-110. [5] Zhu JH, Yang F. Biological process for butanol production. In: Cheng J, editor. Biomass to renewable energy processes. New York: CRC Press: 2009. p. 271-336. [6] Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS. Fermentative butanol production by Clostridia. Biotechnol Bioeng 2008;101:209– 228. [7] Bellodo C, Infante C, Coca M, González-Benito G, Lucas S, García-Cubero MT. Efficient acetone-butanol-ethanol production by Clostridium beijerinckii from sugar beet pulp. Bioresour Technol 2015;190:332-338. [8] Cheng HH, Whang LM, Chan KC, Chung MC, Wu SH, Liu CP, Tien SY, Chen SY, Chang JS, Lee WJ. Biological butanol production from microalgae-based biodiesel residues by Clostridium acetobutylicum. Bioresour Technol 2015;184:379-385. [9] Lin DS, Yen HW, Kao WC, Cheng CL, Chen WM, Huang CC, Chang JS. Bio-butanol production from glycerol with Clostridium pasteurianum CH4: the effects of butyrate addition and in situ butanol removal via membrane distillation. Biotechnol Biofuels 2015;8:168-179. [10] Forsberg CW. Production of 1,3-propanediol from glycerol by Clostridium acetobutylicum and other Clostridium species. Appl Environ Microbiol 1987;53:639-643. [11] Gungormusler M, Gonen C, Azbar N. 1,3-Propanediol production potential by a locally isolated strain of Klebsiella pneumonia in comparison to Clostridium beijerinckii NRRL B593 from waste glycerol. J Polym Environ 2011;19:812-817. [12] Qureshi N, Blaschek HP. Butanol recovery from model solution/fermentation broth by pervaporation: evaluation of membrane performance. Biomass Bioenergy 1999;17:175–184. [13] Miller GL. Use of dinitrosaIicyIic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–428. [14] Hong J. Yield Coefficients for Cell Mass and Product Formation. Biotechnol Bioeng 1989;33:506–507. [15] Yazdani SS, Gonzalez R. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 2007;18:213-219. [16] Quilaguy-Ayure DM, Montoya-Solano JD, Suárez-Moreno ZR, Bernal-Morales JM, Montoya-Castaño D. Analysing the dhaT gene in Colombian Clostridium sp. (Clostridia) 1,3-propanediol-producing strains. Univ Sci 2010;15:17-26.