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Advanced Biomass Value: Microalgae biomass as a new source of sustainable aviation biofuels and lubricant production
New Biotechnology · Volume 31S · July 2014
BIOFUELS, BIOCHEMICALS AND BIOENERGY
Biofuels, biochemicals and bioenergy PB-01 Minimization of Bacterial Contamination with High Solid Loading during Ethanol Production from Lignocellulosic Materials Mofoluwake Taherzadeh 2 1
2
Ishola 1,∗ , Tomas
Brandberg 2 , Mohammad
Swedish Centre for Resource Recovery, University of Borås University of Borås
Abstract Ethanol is the most important renewable fuel in the transportation sector. Its production from lignocellulosic materials, commonly referred to as second generation ethanol, is considered more attractive than production from starch and sugar crops. Bacterial contamination by lactic acid-producing bacteria is still a major problem during ethanol production processes. Bacteria compete with the yeast by consuming the sugars and the nutrients required by the yeast for efficient ethanol production. This often causes substantial economic losses at industrial fermentations. In this study, without any sterilization of the substrate, simultaneous saccharification and fermentation (SSF) was performed using cellulase Cellic® Ctec2 enzyme for hydrolysis and Baker’s yeast, Saccharomyces cerevisiae, was used as the fermenting organism with different loads of suspended solids - 8%, 10% and 12%. With 8% and 10% SS, there was a significant contamination, which caused consumption of both hexoses pentose sugars in the fermentation medium, this resulted in lactic acid concentrations of 43 g/L and 36 g/L from 10% SS and 8% SS respectively. In contrast, only 2.9 g/L lactic acid was observed with 12% SS. An ethanol concentration of 47 g/L was produced from high solid loading of 12% SS while just 26 g/L and 23 g/L were produced from 10% and 8% SS respectively. Our results show that SSF with 12% SS has an increased concentration of inhibitors, particularly acetic acid which selectively inhibited the bacterial growth without affecting the metabolic activities of the yeast during the fermentation Keywords Bacterial contamination; Lignocellulosic ethanol; Saccharomyces cerevisiae, lactic acid. http://dx.doi.org/10.1016/j.nbt.2014.05.1828
PB-02 Breeding low temperature resistant Camelina sativa for biofuel production Florentina Jurcoane 2
Matei 1,∗ , Florentina
Sauca 2 , Paul
Dobre 3 , Stefana
1 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Biotechnologies 2 Centre of Microbial Biotechnology Biotehgen, Bucharest, Romania 3 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Agriculture, Bucharest, Romania
In the European effort to produce sustainable aviation fuel our team is involved in a FP7 project (ITAKA) in breeding activities of Camelina sativa L. as main feedstock. One of our purposes it
is to obtain a variety with high productivity and resistant to low temperatures specific to continental winters as can occur in Romania. This variety may be seeded as autumn culture under minimal tilling conditions. As starting material have been used a local camelina cultivar resistant to low temperatures and an international variety GP202 with high productivity and oil content. It has been taken into account classical approach, the use of immature embryo rescue technique. The first hybrid generation was obtained by castration and pollination; the immature embryos were cultivated in MS medium w/o hormones; new plantlets were cultivated till full maturity under greenhouses conditions. The obtained seeds have been used in open field for other randomized hybridization. The new hybrid registered the following characteristics: the plant lengths and the branching level are non-significantly different compared to the parental lines (ANOVA tests); in terms of productivity, GP202 variety kept its top position, while the new hybrid has proven top position for oil content (2.13% higher in total fat). Regarding the winter resistance, the hybrid has registered 10% more survived plants in open land. As a remark, during the experiments GP202 showed the highest sensibility to the mildew attack. The further aim of the work is to fix the new characters in order to homologate the new variety. http://dx.doi.org/10.1016/j.nbt.2014.05.1829
PB-03 Advanced Biomass Value: Microalgae biomass as a new source of sustainable aviation biofuels and lubricant production Felix Bracharz ∗ , Jan Lorenzen, Farah Qoura TUM, Industrial Biocatalysis
The aviation industry grows 5% p.a. and by 2020 has to comply with strict governmental emission standards that mandate a 20% CO2 reduction compared to the emission levels measured at 2005. Together with the eminent end of fossil resources these drivers force the development of sustainable aviation fuel alternatives that are carbon neutral and in compliance fuel standard regulations such as Jet A. In this study a new, mass-and energy efficient algae biorefinery concept for the integrated production of aviation fuels, industrial lubricants and CO2 adsorbing building materials has been developed. The process chain is based on production of fast growing microalgae biomass containing up to 20%w/w lipids. Subsequently, algae lipids are separated from the biomass fraction and converted to high performance, high value lubricants by targeted functionalisation using a cascade of optimized biocatalytic processes. The biomass residue is enzymatically hydrolysed and used as a fermentation substrate for oleaginous yeast strains. Since these fast growing, oleaginous yeasts can accumulate up to 80% w/w lipids, they are the ideal biomass base for the production of drop-in aviation fuels. Conversion of wet yeast biomass is accomplished using a streamlined thermochemical process that features optimized heterogenous catalysts. In this process carbon rich coke is a residue of the thermochemical biomass processing. This residue stream is used as a settling modifier in the production www.elsevier.com/locate/nbt S93
New Biotechnology · Volume 31S · July 2014
BIOFUELS, BIOCHEMICALS AND BIOENERGY
of CO2 adsorbing building materials. The integrated biorefinery process does not produce any waste streams and adds value to every process intermediate. http://dx.doi.org/10.1016/j.nbt.2014.05.1830
PB-05 The development of biodiesel production using lipase from mutant and selected yeast Aree Rittiboon ∗ , Wannisa Pansuk, Marisa Jatupornpiput King Mongkot’ s Institute of Technology, Ladkrabang
PB-04 Enzymatic production of biodiesel that avoids glycerol as byproduct, by using immobilized Rhizopus Oryzae lipase Carlos Luna 1,∗ , Cristobal Verdugo 2 , Enrique D. Sancho 3 , Diego Luna 4 , Juan Calero 4 , Alejandro Posadillo 5 , Felipa M. Bautista 4 , Antonio A. Romero 4 1
University of Cordoba Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, CSIC 3 Department of Microbiology, University of Cordoba 4 Department of Organic Chemistry, University of Cordoba 5 Seneca Green Catalyst S.L 2
Immobilized Rhizopus oryzae lipase (ROL) was used as biocatalyst on different supports in the selective transesterification reaction of sunflower oil with ethanol to generate a new second generation biodiesel. As ROL it was applied a low cost powdered enzyme preparation from Biocon®-Spain (BIOLIPASE-R), a multipurpose additive used in food industry. In this respect, it was carried out a study to optimize the support used and the immobilization process, as well as the best pH conditions in each case. It has been also evaluated the physical adsorption of this enzyme on a demineralised sepiolite as well as the covalent immobilization of this lipase on amorphous AlPO4 support by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively).On the other hand, the resulting new biofuel, already patented as ECODIESEL® is composed by a mixture of fatty acid ethyl esters and monoacylglycerols (FAEE/MG) blended in a 2/1 molar relation. This novel biofuel, which present the advantage of integrating glycerol as monoacylglycerols (MG) into biofuels composition, exhibits similar physicochemical properties than the conventional biodiesel and it is produced through a process which minimizes waste generation and maximizes efficiency. Finally, one of these immobilization processes of lipases not only was effective with vegetal oils, it has been even successfully carried out the transesterification reaction with animal fat from butchery wastes, using this biocatalyst covalently immobilized with p-hydroxybenzaldehyde, achieving a viable and functional biofuel from low quality raw materials as animal waste. http://dx.doi.org/10.1016/j.nbt.2014.05.1831
S94
www.elsevier.com/locate/nbt
The aim of our research was to select and characterise yeast isolates from soil and other materials produced in the oil palm industry. Of the strains isolated from palm kernels after compression, strain SLP27 produced the highest lipase activity (0.28 unit/ml). Strain SLP27 was mutated sequentially with ultraviolet light for 38 seconds. The resulting strain, UV79, was mutated with ethyl methane sulfonate for 58 minutes to produce strain EM107, which was then mutated with gamma rays at 2 kGy to yield strain GAM47. The maximum lipase activities of these strains were 0.30, 0.36 and 0.70 unit/ml, respectively. Lipase from GAM47 could be used as a catalyst in the transesterification reaction for biodiesel production. This selected strain was identified to species level by analysis of the D1/D2 domain of 26S ribosomal RNA sequence as Candida orthopsilosis. http://dx.doi.org/10.1016/j.nbt.2014.05.1832
PB-06 Production of 3-hydroxybutyrate from waste biomass by metabolically engineered Escherichia coli Johan Jarmander 1,∗ , Mónica Guevara 1 , Mariel Perez Zabaleta 1 , Gustav Sjöberg 1 , Jaroslav Belotserkovsky 1 , Jorge Quillaguamán 2 , Gen Larsson 1 1
Industrial biotechnology, School of biotechnology, KTH Center of biotechnology, Faculty of science and technology, San Simón University
2
There is a vast interest in establishing biorefineries that rely on microbial conversion of biomass for production of fuels and value-added compounds. Especially waste biomasses, such a lignocellulose waste from e.g. agriculture, and municipal food waste are, due to their low cost, attractive substrates for production of biochemicals. Escherichia coli is one of the most commonly used organisms for production of recombinant products since it is well documented, easily manipulated and fast growing in simple media. In this work we aim to use E. coli for production of the chiral compound 3-hydroxybutyrate from waste biomass. This was done by introducing part of the pathway to poly-hydroxybutyrate from the halophilic bacterium Halomonas boliviensis into E. coli. 3-hydroxybutyrate is an industrially important compound as it can be utilized as a drug, as well as in synthesis of various co-polymers. By employing flux modelling and metabolic engineering, the objective is to identify how the internal fluxes and concentrations of metabolites and co-factors can be shifted towards an increased productivity of 3-hydroxybutyrate. This knowledge is also applied in the process design, where limiting one or several elements in the cultivation medium
BIOFUELS, BIOCHEMICALS AND BIOENERGY
Biofuels, biochemicals and bioenergy PB-01 Minimization of Bacterial Contamination with High Solid Loading during Ethanol Production from Lignocellulosic Materials Mofoluwake Taherzadeh 2 1
2
Ishola 1,∗ , Tomas
Brandberg 2 , Mohammad
Swedish Centre for Resource Recovery, University of Borås University of Borås
Abstract Ethanol is the most important renewable fuel in the transportation sector. Its production from lignocellulosic materials, commonly referred to as second generation ethanol, is considered more attractive than production from starch and sugar crops. Bacterial contamination by lactic acid-producing bacteria is still a major problem during ethanol production processes. Bacteria compete with the yeast by consuming the sugars and the nutrients required by the yeast for efficient ethanol production. This often causes substantial economic losses at industrial fermentations. In this study, without any sterilization of the substrate, simultaneous saccharification and fermentation (SSF) was performed using cellulase Cellic® Ctec2 enzyme for hydrolysis and Baker’s yeast, Saccharomyces cerevisiae, was used as the fermenting organism with different loads of suspended solids - 8%, 10% and 12%. With 8% and 10% SS, there was a significant contamination, which caused consumption of both hexoses pentose sugars in the fermentation medium, this resulted in lactic acid concentrations of 43 g/L and 36 g/L from 10% SS and 8% SS respectively. In contrast, only 2.9 g/L lactic acid was observed with 12% SS. An ethanol concentration of 47 g/L was produced from high solid loading of 12% SS while just 26 g/L and 23 g/L were produced from 10% and 8% SS respectively. Our results show that SSF with 12% SS has an increased concentration of inhibitors, particularly acetic acid which selectively inhibited the bacterial growth without affecting the metabolic activities of the yeast during the fermentation Keywords Bacterial contamination; Lignocellulosic ethanol; Saccharomyces cerevisiae, lactic acid. http://dx.doi.org/10.1016/j.nbt.2014.05.1828
PB-02 Breeding low temperature resistant Camelina sativa for biofuel production Florentina Jurcoane 2
Matei 1,∗ , Florentina
Sauca 2 , Paul
Dobre 3 , Stefana
1 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Biotechnologies 2 Centre of Microbial Biotechnology Biotehgen, Bucharest, Romania 3 University of Agronomical Sciences and Veterinary Medicine Bucharest, Faculty of Agriculture, Bucharest, Romania
In the European effort to produce sustainable aviation fuel our team is involved in a FP7 project (ITAKA) in breeding activities of Camelina sativa L. as main feedstock. One of our purposes it
is to obtain a variety with high productivity and resistant to low temperatures specific to continental winters as can occur in Romania. This variety may be seeded as autumn culture under minimal tilling conditions. As starting material have been used a local camelina cultivar resistant to low temperatures and an international variety GP202 with high productivity and oil content. It has been taken into account classical approach, the use of immature embryo rescue technique. The first hybrid generation was obtained by castration and pollination; the immature embryos were cultivated in MS medium w/o hormones; new plantlets were cultivated till full maturity under greenhouses conditions. The obtained seeds have been used in open field for other randomized hybridization. The new hybrid registered the following characteristics: the plant lengths and the branching level are non-significantly different compared to the parental lines (ANOVA tests); in terms of productivity, GP202 variety kept its top position, while the new hybrid has proven top position for oil content (2.13% higher in total fat). Regarding the winter resistance, the hybrid has registered 10% more survived plants in open land. As a remark, during the experiments GP202 showed the highest sensibility to the mildew attack. The further aim of the work is to fix the new characters in order to homologate the new variety. http://dx.doi.org/10.1016/j.nbt.2014.05.1829
PB-03 Advanced Biomass Value: Microalgae biomass as a new source of sustainable aviation biofuels and lubricant production Felix Bracharz ∗ , Jan Lorenzen, Farah Qoura TUM, Industrial Biocatalysis
The aviation industry grows 5% p.a. and by 2020 has to comply with strict governmental emission standards that mandate a 20% CO2 reduction compared to the emission levels measured at 2005. Together with the eminent end of fossil resources these drivers force the development of sustainable aviation fuel alternatives that are carbon neutral and in compliance fuel standard regulations such as Jet A. In this study a new, mass-and energy efficient algae biorefinery concept for the integrated production of aviation fuels, industrial lubricants and CO2 adsorbing building materials has been developed. The process chain is based on production of fast growing microalgae biomass containing up to 20%w/w lipids. Subsequently, algae lipids are separated from the biomass fraction and converted to high performance, high value lubricants by targeted functionalisation using a cascade of optimized biocatalytic processes. The biomass residue is enzymatically hydrolysed and used as a fermentation substrate for oleaginous yeast strains. Since these fast growing, oleaginous yeasts can accumulate up to 80% w/w lipids, they are the ideal biomass base for the production of drop-in aviation fuels. Conversion of wet yeast biomass is accomplished using a streamlined thermochemical process that features optimized heterogenous catalysts. In this process carbon rich coke is a residue of the thermochemical biomass processing. This residue stream is used as a settling modifier in the production www.elsevier.com/locate/nbt S93
New Biotechnology · Volume 31S · July 2014
BIOFUELS, BIOCHEMICALS AND BIOENERGY
of CO2 adsorbing building materials. The integrated biorefinery process does not produce any waste streams and adds value to every process intermediate. http://dx.doi.org/10.1016/j.nbt.2014.05.1830
PB-05 The development of biodiesel production using lipase from mutant and selected yeast Aree Rittiboon ∗ , Wannisa Pansuk, Marisa Jatupornpiput King Mongkot’ s Institute of Technology, Ladkrabang
PB-04 Enzymatic production of biodiesel that avoids glycerol as byproduct, by using immobilized Rhizopus Oryzae lipase Carlos Luna 1,∗ , Cristobal Verdugo 2 , Enrique D. Sancho 3 , Diego Luna 4 , Juan Calero 4 , Alejandro Posadillo 5 , Felipa M. Bautista 4 , Antonio A. Romero 4 1
University of Cordoba Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, CSIC 3 Department of Microbiology, University of Cordoba 4 Department of Organic Chemistry, University of Cordoba 5 Seneca Green Catalyst S.L 2
Immobilized Rhizopus oryzae lipase (ROL) was used as biocatalyst on different supports in the selective transesterification reaction of sunflower oil with ethanol to generate a new second generation biodiesel. As ROL it was applied a low cost powdered enzyme preparation from Biocon®-Spain (BIOLIPASE-R), a multipurpose additive used in food industry. In this respect, it was carried out a study to optimize the support used and the immobilization process, as well as the best pH conditions in each case. It has been also evaluated the physical adsorption of this enzyme on a demineralised sepiolite as well as the covalent immobilization of this lipase on amorphous AlPO4 support by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively).On the other hand, the resulting new biofuel, already patented as ECODIESEL® is composed by a mixture of fatty acid ethyl esters and monoacylglycerols (FAEE/MG) blended in a 2/1 molar relation. This novel biofuel, which present the advantage of integrating glycerol as monoacylglycerols (MG) into biofuels composition, exhibits similar physicochemical properties than the conventional biodiesel and it is produced through a process which minimizes waste generation and maximizes efficiency. Finally, one of these immobilization processes of lipases not only was effective with vegetal oils, it has been even successfully carried out the transesterification reaction with animal fat from butchery wastes, using this biocatalyst covalently immobilized with p-hydroxybenzaldehyde, achieving a viable and functional biofuel from low quality raw materials as animal waste. http://dx.doi.org/10.1016/j.nbt.2014.05.1831
S94
www.elsevier.com/locate/nbt
The aim of our research was to select and characterise yeast isolates from soil and other materials produced in the oil palm industry. Of the strains isolated from palm kernels after compression, strain SLP27 produced the highest lipase activity (0.28 unit/ml). Strain SLP27 was mutated sequentially with ultraviolet light for 38 seconds. The resulting strain, UV79, was mutated with ethyl methane sulfonate for 58 minutes to produce strain EM107, which was then mutated with gamma rays at 2 kGy to yield strain GAM47. The maximum lipase activities of these strains were 0.30, 0.36 and 0.70 unit/ml, respectively. Lipase from GAM47 could be used as a catalyst in the transesterification reaction for biodiesel production. This selected strain was identified to species level by analysis of the D1/D2 domain of 26S ribosomal RNA sequence as Candida orthopsilosis. http://dx.doi.org/10.1016/j.nbt.2014.05.1832
PB-06 Production of 3-hydroxybutyrate from waste biomass by metabolically engineered Escherichia coli Johan Jarmander 1,∗ , Mónica Guevara 1 , Mariel Perez Zabaleta 1 , Gustav Sjöberg 1 , Jaroslav Belotserkovsky 1 , Jorge Quillaguamán 2 , Gen Larsson 1 1
Industrial biotechnology, School of biotechnology, KTH Center of biotechnology, Faculty of science and technology, San Simón University
2
There is a vast interest in establishing biorefineries that rely on microbial conversion of biomass for production of fuels and value-added compounds. Especially waste biomasses, such a lignocellulose waste from e.g. agriculture, and municipal food waste are, due to their low cost, attractive substrates for production of biochemicals. Escherichia coli is one of the most commonly used organisms for production of recombinant products since it is well documented, easily manipulated and fast growing in simple media. In this work we aim to use E. coli for production of the chiral compound 3-hydroxybutyrate from waste biomass. This was done by introducing part of the pathway to poly-hydroxybutyrate from the halophilic bacterium Halomonas boliviensis into E. coli. 3-hydroxybutyrate is an industrially important compound as it can be utilized as a drug, as well as in synthesis of various co-polymers. By employing flux modelling and metabolic engineering, the objective is to identify how the internal fluxes and concentrations of metabolites and co-factors can be shifted towards an increased productivity of 3-hydroxybutyrate. This knowledge is also applied in the process design, where limiting one or several elements in the cultivation medium