Bioresource Technology 159 (2014) 167–175
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production Afroditi Chatzifragkou a,1, Seraphim Papanikolaou a, Nikolaos Kopsahelis a, Vasiliki Kachrimanidou a, Maria Pilar Dorado b, Apostolis A. Koutinas a,⇑ a
Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece Department of Physical Chemistry and Applied Thermodynamics, Escuela Politecnica Superior, University of Cordoba, Campus de Excelencia Agroalimentario, ceiA3, 23071 Cordoba, Spain b
h i g h l i g h t s Rapeseed meal (RSM) hydrolysate was evaluated as nutrient for C. butyricum growth. Free amino nitrogen concentrations of RSM hydrolysates influenced PDO production. RSM hydrolysate effectively replaced the need of expensive nitrogen sources. Repeated batch cultures with RSM hydrolysate enhanced PDO production (65.5 g/L). RSM hydrolysate suitability in PDO fermentation is demonstrated for the first time.
a r t i c l e
i n f o
Article history: Received 3 October 2013 Received in revised form 6 February 2014 Accepted 8 February 2014 Available online 15 February 2014 Keywords: Biorefineries Rapeseed meal hydrolysate Crude glycerol 1,3-Propanediol Clostridium butyricum
a b s t r a c t Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The international rising demand on renewable fuels is driven by environmental concerns, depleting petroleum resources and public awareness. As a result, biodiesel production has been noticeably increased during the last decade (Papanikolaou, 2009). The two major by-products of oilseed-based biodiesel production processes ⇑ Corresponding author. Address: Laboratory of Food Process Engineering, Processing and Preservation of Agricultural Products, Department of Food Science and Human Nutrition, Agricultural University of Athens, Greece. Tel./fax: +30 210 5294729. E-mail address: [email protected] (A.A. Koutinas). 1 Present address: Department of Food and Nutritional Sciences, University of Reading, Whiteknights, Reading RG6 6AD, UK. http://dx.doi.org/10.1016/j.biortech.2014.02.021 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
are crude glycerol and oilseed cakes or meals (Koutinas et al., 2007; Lomascolo et al., 2012). Oilseed meals represent by-product streams remaining after oil extraction from oilseeds, such as rapeseed, soybean and sunflower. Glycerol is generated as a 10% (w/w) by-product during trans-esterification of triacylglycerols into fatty acid alkyl esters in the presence of alcohol (Chatzifragkou and Papanikolaou, 2012; Lomascolo et al., 2012). The composition of glycerol-rich streams generated by biodiesel producers, called crude glycerol, contain variable composition of glycerol (77–90%), water (5.3–14.2%), methanol (up to 1.7%), residual fatty acids and corresponding esters, and either NaCl (4.2–5.5%) or K2SO4 (0.8–6.6%) depending on the catalyst used during transesterification (Mothes et al., 2007; Chatzifragkou and Papanikolaou, 2012). The development of advanced oilseed-based biorefineries is dependent
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A. Chatzifragkou et al. / Bioresource Technology 159 (2014) 167–175
on the efficient valorization of these two by-product streams as renewable feedstocks for the production of a wide range of products, such as chemicals, biomaterials, high-value products and energy (Koutinas et al., 2007). Rapeseed meal (RSM) is principally utilized as combustion material, organic fertilizer and to a lesser extent as a livestock feed (Nitayavardhana and Khanal, 2012). RSM contains protein (34–36%, w/w), carbohydrates, as well as minerals, such as calcium, phosphorus and iron (Lomascolo et al., 2012; Wang et al., 2010). The production of fermentation nutrient supplements from RSM necessitates the application of enzymatic pre-treatment of RSM, since the available protein content is hardly assimilable by the majority of bacterial and yeast strains (Wang et al., 2010). This can be achieved through the utilization of fungi in solid state fermentations, such as Aspergillus oryzae, a mold characterized as efficient producer of predominantly protease and, to a lesser extent, of phytase, cellulase, xylanase, as well as amylolytic enzymes. As a consequence, the produced hydrolysates contain amino acids, peptides, phosphorus and low amounts of reducing sugars, representing, thus, a rich-nutrient source for microbial fermentations (Koutinas et al., 2007; Wang et al., 2010). The potential of RSM hydrolysate as feedstock for microbial bioconversions has only recently been validated by a scarce number of studies. Wang et al. (2010) reported that RSM hydrolysates provided all essential nutrients (with the exception of carbon source) required for Saccharomyces cerevisiae growth, while nutritional similarities between the produced feedstock and a mixture of yeast extract and peptone were verified. RSM hydrolysate has been successfully utilized for microbial oil production via fed-batch cultures using the oleaginous yeast strain Rhodosporidium toruloides Y4 (Kiran et al., 2012). Garcia et al. (2013) reported the utilisation of RSM hydrolysate as nutrient supplement for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers during shake flask cultures carried out with the bacterial strain Curpriavidus necator DSM 545. As far as crude glycerol is concerned, its biotransformation into (high) added-value products through microbial fermentations is considered as one of the most promising valorization means (Xiu and Zeng, 2008). A variety of prokaryotic and eukaryotic microorganisms are known for their ability to assimilate glycerol as the sole or supplementary carbon source and synthesize a plethora of metabolic products, such as organic acids (André et al., 2010; Vlysidis et al., 2011), 1,3-propanediol (Chatzifragkou et al., 2011a; Hirschmann et al., 2005; Wilkens et al., 2012), single cell oils (Papanikolaou and Aggelis, 2002), ethanol (Metsoviti et al., 2012), 2,3-butanediol (Metsoviti et al., 2012) and polyhydroxyalkanoates (Kachrimanidou et al., 2013). Among those, 1,3-propanediol (PDO) has currently re-gained considerable attention, due to its recognition as a green platform chemical with a wide spectrum of industrial applications (Zeng and Sabra, 2011). Moreover, the launch of a PDO-based polyester, namely polymethylene terephthalate termed PTT (Shell) or 3GT (Dupont), with a number of excellent physical properties, has attracted much interest in the field of novel polymer production (Chatzifragkou and Papanikolaou, 2012; Zeng and Sabra, 2011). This study has evaluated the potential of integrating PDO production in rapeseed-based biodiesel plants using entirely RSM meal and crude glycerol for the production of nutrient-complete fermentation media. The microbial strain used in this study is an efficient produced of PDO (70.8 g/L) when crude glycerol is supplemented with commercial nutrient supplements (Chatzifragkou et al., 2011b). Therefore, the main goal of this study was the investigation of the potential to replace commercial nutrient supplements by RSM hydrolysates. The rationale followed involved the pre-treatment of RSM through enzymatic hydrolysis utilizing enzyme consortia obtained via solid state fermentation. The
nutrient-rich hydrolysate, supplemented with crude glycerol, was then evaluated as a suitable fermentation feedstock in PDO fermentations. 2. Methods 2.1. Microorganisms An industrial fungal strain of A. oryzae isolated from a soy sauce starter at the company Amoy Food Ltd. (Hong Kong), kindly provided by Professor Colin Webb (Satake Centre for Grain Process Engineering, University of Manchester, UK), was employed in solid state fermentations (SSF) to produce crude enzymes essential for RSM hydrolysis. The strain was maintained in the form of spores, dry in sand at 4 °C. For the preparation of inoculum for SSF, spores were purified and sporulated in slopes, in a solid medium containing 25 g/L RSM, 25 g/L wheat bran and 20 g/L agar. The bacterial strain C. butyricum VPI 1718 (Chatzifragkou et al., 2011a) was used to evaluate the suitability of the feedstock derived from RSM for PDO fermentation. The microorganism was maintained in Reinforced Clostridial Medium (RCM), in 50 mL anaerobic flasks at 4 °C. Before any experiment, a heat-shock (80 °C/10 min) was performed, in order to stimulate the germination of the spores. Then, precultures of the post-germination culture were performed in 200 mL anaerobic flasks filled with 50 mL of preculture medium, as described by Chatzifragkou et al. (2011b), infused with nitrogen gas and incubated at 35 °C for 12 h. 2.2. Raw materials Biodiesel-derived crude glycerol kindly provided by ‘‘ADM Industries’’ (Hamburg, Germany), originated from rapeseed oil trans-esterification, was used in various initial concentrations as carbon source of the bacterial fermentations. The aforementioned raw material had the following composition (w/w): glycerol, 81%; water, 10–12%; potassium salts, 5.0–7.0%; free-fatty acids, 1.0% and methanol,
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production Afroditi Chatzifragkou a,1, Seraphim Papanikolaou a, Nikolaos Kopsahelis a, Vasiliki Kachrimanidou a, Maria Pilar Dorado b, Apostolis A. Koutinas a,⇑ a
Department of Food Science and Human Nutrition, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece Department of Physical Chemistry and Applied Thermodynamics, Escuela Politecnica Superior, University of Cordoba, Campus de Excelencia Agroalimentario, ceiA3, 23071 Cordoba, Spain b
h i g h l i g h t s Rapeseed meal (RSM) hydrolysate was evaluated as nutrient for C. butyricum growth. Free amino nitrogen concentrations of RSM hydrolysates influenced PDO production. RSM hydrolysate effectively replaced the need of expensive nitrogen sources. Repeated batch cultures with RSM hydrolysate enhanced PDO production (65.5 g/L). RSM hydrolysate suitability in PDO fermentation is demonstrated for the first time.
a r t i c l e
i n f o
Article history: Received 3 October 2013 Received in revised form 6 February 2014 Accepted 8 February 2014 Available online 15 February 2014 Keywords: Biorefineries Rapeseed meal hydrolysate Crude glycerol 1,3-Propanediol Clostridium butyricum
a b s t r a c t Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The international rising demand on renewable fuels is driven by environmental concerns, depleting petroleum resources and public awareness. As a result, biodiesel production has been noticeably increased during the last decade (Papanikolaou, 2009). The two major by-products of oilseed-based biodiesel production processes ⇑ Corresponding author. Address: Laboratory of Food Process Engineering, Processing and Preservation of Agricultural Products, Department of Food Science and Human Nutrition, Agricultural University of Athens, Greece. Tel./fax: +30 210 5294729. E-mail address: [email protected] (A.A. Koutinas). 1 Present address: Department of Food and Nutritional Sciences, University of Reading, Whiteknights, Reading RG6 6AD, UK. http://dx.doi.org/10.1016/j.biortech.2014.02.021 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
are crude glycerol and oilseed cakes or meals (Koutinas et al., 2007; Lomascolo et al., 2012). Oilseed meals represent by-product streams remaining after oil extraction from oilseeds, such as rapeseed, soybean and sunflower. Glycerol is generated as a 10% (w/w) by-product during trans-esterification of triacylglycerols into fatty acid alkyl esters in the presence of alcohol (Chatzifragkou and Papanikolaou, 2012; Lomascolo et al., 2012). The composition of glycerol-rich streams generated by biodiesel producers, called crude glycerol, contain variable composition of glycerol (77–90%), water (5.3–14.2%), methanol (up to 1.7%), residual fatty acids and corresponding esters, and either NaCl (4.2–5.5%) or K2SO4 (0.8–6.6%) depending on the catalyst used during transesterification (Mothes et al., 2007; Chatzifragkou and Papanikolaou, 2012). The development of advanced oilseed-based biorefineries is dependent
168
A. Chatzifragkou et al. / Bioresource Technology 159 (2014) 167–175
on the efficient valorization of these two by-product streams as renewable feedstocks for the production of a wide range of products, such as chemicals, biomaterials, high-value products and energy (Koutinas et al., 2007). Rapeseed meal (RSM) is principally utilized as combustion material, organic fertilizer and to a lesser extent as a livestock feed (Nitayavardhana and Khanal, 2012). RSM contains protein (34–36%, w/w), carbohydrates, as well as minerals, such as calcium, phosphorus and iron (Lomascolo et al., 2012; Wang et al., 2010). The production of fermentation nutrient supplements from RSM necessitates the application of enzymatic pre-treatment of RSM, since the available protein content is hardly assimilable by the majority of bacterial and yeast strains (Wang et al., 2010). This can be achieved through the utilization of fungi in solid state fermentations, such as Aspergillus oryzae, a mold characterized as efficient producer of predominantly protease and, to a lesser extent, of phytase, cellulase, xylanase, as well as amylolytic enzymes. As a consequence, the produced hydrolysates contain amino acids, peptides, phosphorus and low amounts of reducing sugars, representing, thus, a rich-nutrient source for microbial fermentations (Koutinas et al., 2007; Wang et al., 2010). The potential of RSM hydrolysate as feedstock for microbial bioconversions has only recently been validated by a scarce number of studies. Wang et al. (2010) reported that RSM hydrolysates provided all essential nutrients (with the exception of carbon source) required for Saccharomyces cerevisiae growth, while nutritional similarities between the produced feedstock and a mixture of yeast extract and peptone were verified. RSM hydrolysate has been successfully utilized for microbial oil production via fed-batch cultures using the oleaginous yeast strain Rhodosporidium toruloides Y4 (Kiran et al., 2012). Garcia et al. (2013) reported the utilisation of RSM hydrolysate as nutrient supplement for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers during shake flask cultures carried out with the bacterial strain Curpriavidus necator DSM 545. As far as crude glycerol is concerned, its biotransformation into (high) added-value products through microbial fermentations is considered as one of the most promising valorization means (Xiu and Zeng, 2008). A variety of prokaryotic and eukaryotic microorganisms are known for their ability to assimilate glycerol as the sole or supplementary carbon source and synthesize a plethora of metabolic products, such as organic acids (André et al., 2010; Vlysidis et al., 2011), 1,3-propanediol (Chatzifragkou et al., 2011a; Hirschmann et al., 2005; Wilkens et al., 2012), single cell oils (Papanikolaou and Aggelis, 2002), ethanol (Metsoviti et al., 2012), 2,3-butanediol (Metsoviti et al., 2012) and polyhydroxyalkanoates (Kachrimanidou et al., 2013). Among those, 1,3-propanediol (PDO) has currently re-gained considerable attention, due to its recognition as a green platform chemical with a wide spectrum of industrial applications (Zeng and Sabra, 2011). Moreover, the launch of a PDO-based polyester, namely polymethylene terephthalate termed PTT (Shell) or 3GT (Dupont), with a number of excellent physical properties, has attracted much interest in the field of novel polymer production (Chatzifragkou and Papanikolaou, 2012; Zeng and Sabra, 2011). This study has evaluated the potential of integrating PDO production in rapeseed-based biodiesel plants using entirely RSM meal and crude glycerol for the production of nutrient-complete fermentation media. The microbial strain used in this study is an efficient produced of PDO (70.8 g/L) when crude glycerol is supplemented with commercial nutrient supplements (Chatzifragkou et al., 2011b). Therefore, the main goal of this study was the investigation of the potential to replace commercial nutrient supplements by RSM hydrolysates. The rationale followed involved the pre-treatment of RSM through enzymatic hydrolysis utilizing enzyme consortia obtained via solid state fermentation. The
nutrient-rich hydrolysate, supplemented with crude glycerol, was then evaluated as a suitable fermentation feedstock in PDO fermentations. 2. Methods 2.1. Microorganisms An industrial fungal strain of A. oryzae isolated from a soy sauce starter at the company Amoy Food Ltd. (Hong Kong), kindly provided by Professor Colin Webb (Satake Centre for Grain Process Engineering, University of Manchester, UK), was employed in solid state fermentations (SSF) to produce crude enzymes essential for RSM hydrolysis. The strain was maintained in the form of spores, dry in sand at 4 °C. For the preparation of inoculum for SSF, spores were purified and sporulated in slopes, in a solid medium containing 25 g/L RSM, 25 g/L wheat bran and 20 g/L agar. The bacterial strain C. butyricum VPI 1718 (Chatzifragkou et al., 2011a) was used to evaluate the suitability of the feedstock derived from RSM for PDO fermentation. The microorganism was maintained in Reinforced Clostridial Medium (RCM), in 50 mL anaerobic flasks at 4 °C. Before any experiment, a heat-shock (80 °C/10 min) was performed, in order to stimulate the germination of the spores. Then, precultures of the post-germination culture were performed in 200 mL anaerobic flasks filled with 50 mL of preculture medium, as described by Chatzifragkou et al. (2011b), infused with nitrogen gas and incubated at 35 °C for 12 h. 2.2. Raw materials Biodiesel-derived crude glycerol kindly provided by ‘‘ADM Industries’’ (Hamburg, Germany), originated from rapeseed oil trans-esterification, was used in various initial concentrations as carbon source of the bacterial fermentations. The aforementioned raw material had the following composition (w/w): glycerol, 81%; water, 10–12%; potassium salts, 5.0–7.0%; free-fatty acids, 1.0% and methanol,
