Processes for the biotechnological production of kerosene and diesel blendstocks are often econom... more Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a b...
There has recently been a renewed interest in converting the carbohydrate content (cellulose and ... more There has recently been a renewed interest in converting the carbohydrate content (cellulose and hemicellulose) of lignocellulosic biomass to biofuels and biochemicals. This conversion requires depolymerization of the carbohydrate content of biomass to fermentable monosaccharides by using glycoside hydrolases (GHs). Despite the vast number of GHs deposited in publicly available databases, 90 % of these enzymes are not characterized and many are unlikely to be suitable for industrial applications because of low activity and stability under processing conditions. While protein engineering (by using rational strategies as well as directed evolution) has been used to evolve GHs for industrial applications, bioprospecting and metagenomics provide alternative approaches to finding GHs with desired properties. The identification of new GHs from large genetic inventories relies on efficient protein expression and rapid high-throughput screening or selection to evaluate large enzyme libraries. However, heterologous expression of GHs (especially cellulases) in microbial hosts is usually very difficult, and often results in misfolded and/or inactive enzymes or non-native enzyme structures. Furthermore, the poor correlation between the activity of many GHs on soluble and insoluble substrates necessitates the highthroughput screening to be carried out on insoluble lignocellulosic substrates. Herein, we present the development of a one-pot high-throughput in vitro glycoside hydrolase (HIGH) expression and screening method by using insoluble lignocellulosic substrates. The HIGH platform can be adapted to screen for cellulases, xylanases, amylases, and b-glucosidases.
The attractiveness of fermentor cultures of Dioscorea deltoidea Wall. (Dioscoreaceae) as a source... more The attractiveness of fermentor cultures of Dioscorea deltoidea Wall. (Dioscoreaceae) as a source of diosgenin has been greatly improved by switching away from the traditional product recovery method that has been used in all previous studies. By using a known but little-used ...
Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge ... more Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge Tethya californiana was analyzed based on 16S rRNA gene sequences. One candidate division and nine bacterial phyla were detected, including members of all five subdivisions of Proteobacteria. Moreover, chloroplast-derived Stramenopiles- and Rhodophyta-affiliated 16S rRNA gene sequences were found and Stramenopiles represented the most abundant clones (30%) in the clone library. On the phylum-level, the microbial fingerprint of T. californiana showed a similar pattern as its Mediterranean relative T. aurantium. An interesting difference was that Cyanobacteria that were abundantly present in T. aurantium were not found in T. californiana, but that the latter sponges harbored phototrophic Stramenopiles instead. Surprisingly, the phototrophic microorganisms were evenly distributed over the inner and outer parts of the sponge tissue, which implies that they also reside in regions without dire...
Closely related to the "protein folding problem" is the issue of protein misfolding and... more Closely related to the "protein folding problem" is the issue of protein misfolding and aggregation. Protein aggregation has been associated with the pathologies of nearly 20 human diseases and presents serious difficulties during the manufacture of pharmaceutical proteins. Computational studies of multiprotein systems have recently emerged as a powerful complement to experimental efforts aimed at understanding the mechanisms of protein aggregation. We describe the thermodynamics of systems containing two lattice-model 64-mers. A parallel tempering algorithm abates problems associated with glassy systems and the weighted histogram analysis method improves statistical quality. The presence of a second chain has a substantial effect on single-chain conformational preferences. The melting temperature is substantially reduced, and the increase in the population of unfolded states is correlated with an increase in interactions between chains. The transition from two native chai...
Protein aggregation has been associated with a number of human diseases, and is a serious problem... more Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregatio...
Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the ex... more Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the experimental mixed-solute-exclusion method, MSE (introduced by Kuga), to obtain the solute-exclusion curve representing the amount of imbibed liquid inside the gel inaccessible for a solute of radius r. We use the Brownian motion model (developed by Cassasa) to convert the size-exclusion curve into the pore-size distribution, which gives the frequency of pore radius R as a function of R. This theoretically-based interpretation of MSE data leads to the Fredholm integral equation that we solve numerically. Results are reported for a series of hydrogels containing acrylamide and 3 % MAPTAC; the hydrogels differed in extent of cross-linking and/or initial concentration of monomer. Pore-size distributions shift to lower pore sizes with rising initial monomer concentration and with rising cross-linker-to-monomer ratio.
Background: Ionic liquid (IL) pretreatment could enable an economically viable route to produce b... more Background: Ionic liquid (IL) pretreatment could enable an economically viable route to produce biofuels by providing efficient means to extract sugars and lignin from lignocellulosic biomass. However, to realize this, novel IL-based processes need to be developed in order to minimize the overall production costs and accelerate commercial viability. In this study, two variants of IL-based processes are considered: one based on complete removal of the IL prior to hydrolysis using a water-wash (WW) step and the other based on a "one-pot" (OP) process that does not require IL removal prior to saccharification. Detailed techno-economic analysis (TEA) of these two routes was carried out to understand the cost drivers, economic potential (minimum ethanol selling price, MESP), and relative merits and challenges of each route. Results: At high biomass loading (50%), both routes exhibited comparable economic performance with an MESP of $6.3/gal. With the possible advances identified (reduced water or acid/base consumption, improved conversion in pretreatment, and lignin valorization), the MESP could be reduced to around $3/gal ($3.2 in the WW route and $2.8 in the OP route). Conclusions: It was found that, to be competitive at industrial scale, lowered cost of ILs used and higher biomass loadings (50%) are essential for both routes, and in particular for the OP route. Overall, while the economic potential of both routes appears to be comparable at higher biomass loadings, the OP route showed the benefit of lower water consumption at the plant level, an important cost and sustainability consideration for biorefineries.
Background: Trichoderma reesei is a key cellulase source for economically saccharifying cellulosi... more Background: Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60-70°C. Results: A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50-70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T m ) and improved half-life at 60°C (t 1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t 1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes. Conclusion: Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.
Clostridium acetobutylicum is a bacterial species that ferments sugar to a mixture of organic sol... more Clostridium acetobutylicum is a bacterial species that ferments sugar to a mixture of organic solvents (acetone, butanol and ethanol). This protocol delineates a methodology to combine solventogenic clostridial fermentation and chemical catalysis via extractive fermentation for the production of biofuel blendstocks. Extractive fermentation of C. acetobutylicum is operated in fed-batch mode with a concentrated feed solution (500 grams per liter glucose and 50 grams per liter yeast extract) for 60 h, producing in excess of 40 g of solvents (acetone, butanol and ethanol) between the completely immiscible extractant and aqueous phases of the bioreactor. After distillation of the extractant phase, the acetone, butanol and ethanol mixture is upgraded to long-chain ketones over a palladium-hydrotalcite (Pd-HT) catalyst. This reaction is generally carried out in batch with a high-pressure Q-tube for 20 h at 250 °C. Following this protocol enables the production of ∼0.5 g of high-value biofu...
A large number of novel compounds with significant medical potential have been isolated from spon... more A large number of novel compounds with significant medical potential have been isolated from sponges, motivating efforts to develop techniques for the sustainable cultivation of sponge biomass. To date, 33 sponges from nine different orders have been examined to assess their ability to be cultured in vitro. However, little consideration has been given to the relationships between these sponges; only one report has considering the phylogenetic relationships between the species. On the basis of morphological data, no taxonomic specificity was apparent as an indicator for the successful cultivation of the sponges. As the systematic classification of the Demospongiae is poorly understood, we collated available information on the success of in vitro sponge cell cultivation reports and examined the phylogenetic relationships of these sponges through the use of 18S and 28S rDNA sequence data. Based on molecular data, the ability of sponges to form primary aggregates from the dissociated cells of marine demosponges indicates that taxonomic trends may exist, emphasizing the need to better characterize sponges being investigated for biotechnological applications.
Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the ex... more Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the experimental mixed-solute-exclusion method, MSE (introduced by Kuga), to obtain the solute-exclusion curve representing the amount of imbibed liquid inside the gel inaccessible for a solute of radius r. We use the Brownian motion model (developed by Cassasa) to convert the size-exclusion curve into the pore-size distribution, which gives the frequency of pore radius R as a function of R. This theoretically-based interpretation of MSE data leads to the Fredholm integral equation that we solve numerically. Results are reported for a series of hydrogels containing acrylamide and 3 % MAPTAC; the hydrogels differed in extent of cross-linking and/or initial concentration of monomer. Pore-size distributions shift to lower pore sizes with rising initial monomer concentration and with rising cross-linker-to-monomer ratio.
Sponges have rich and diverse associated microbial communities, which may have important function... more Sponges have rich and diverse associated microbial communities, which may have important functions in their metabolism. A survey of the archaeal communities of 23 poriferan species, focusing on the family Axinellidae, was conducted over the period 2002-2004 using 16S rDNA gene libraries created with archaeal-speciWc primers. The 28S rDNA sequences of the sponge hosts were also obtained. Of 23 species, 19 showed evidence of archaeal communities from group C1a (marine group I; Crenarchaeota), with three of these also showing evidence of Archaea from group E2 (marine group II; Euryarchaeota). Within the Crenarchaeota, two strongly supported sponge-speciWc clades were identiWed corresponding to the sponge family Axinellidae, and a novel sponge clade denoted clade C. These Wndings suggest that these archaea have evolved closely with their sponge hosts and are likely to play an important role in their metabolism.
Protein aggregation has been associated with a number of human diseases, and is a serious problem... more Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregation, although most association events take place at times much shorter than those required for folding. Last, we discuss results presented here as a basis for future work aimed at rational design of mutations to reduce aggregation propensity, as well as for development of small-molecular weight refolding enhancers. B 2004 Wiley Periodicals, Inc.
The addition of a biorefinery to an organic farm was investigated, where ethanol was produced fro... more The addition of a biorefinery to an organic farm was investigated, where ethanol was produced from germinated rye grains and whey, and the effluent was separated into two streams: the protein-rich solid fraction, to be used as animal feed, and the liquid fraction, which can be co-digested with clover grass silage to produce biogas. A method for ethanol production from rye was applied by utilizing inherent amylase activity from germination of the seed. Biogas potential of ethanol fermentation effluent was measured through anaerobic digestion trials. The effluent from the trials was assumed to serve as natural fertilizer. A technoeconomic analysis was also performed; total capital investment was estimated to be approximately 4 M USD. Setting a methane selling price according to available incentives for ''green electricity'' (0.72 USD/m 3 ) led to a minimum ethanol selling price of 1.89 USD/L (project lifetime 25 yr, at a discount rate 10%).
ABSTRACT This chapter contains sections titled: IntroductionProduction ProcessesBiomass Transport... more ABSTRACT This chapter contains sections titled: IntroductionProduction ProcessesBiomass Transportation and HandlingConversion of Biomass into SugarsConversion of Sugars into BiofuelsSeparation and PurificationCo-product HandlingMajor Cost DriversRisksPolicy SupportInfrastructure and Vehicle ModificationsConclusions Acknowledgments
Processes for the biotechnological production of kerosene and diesel blendstocks are often econom... more Processes for the biotechnological production of kerosene and diesel blendstocks are often economically unattractive due to low yields and product titers. Recently, Clostridium acetobutylicum fermentation products acetone, butanol, and ethanol (ABE) were shown to serve as precursors for catalytic upgrading to higher chain-length molecules that can be used as fuel substitutes. To produce suitable kerosene and diesel blendstocks, the butanol:acetone ratio of fermentation products needs to be increased to 2-2.5:1, while ethanol production is minimized. Here we show that the overexpression of selected proteins changes the ratio of ABE products relative to the wild type ATCC 824 strain. Overexpression of the native alcohol/aldehyde dehydrogenase (AAD) has been reported to primarily increase ethanol formation in C. acetobutylicum. We found that overexpression of the AAD(D485G) variant increased ethanol titers by 294%. Catalytic upgrading of the 824(aad(D485G)) ABE products resulted in a b...
There has recently been a renewed interest in converting the carbohydrate content (cellulose and ... more There has recently been a renewed interest in converting the carbohydrate content (cellulose and hemicellulose) of lignocellulosic biomass to biofuels and biochemicals. This conversion requires depolymerization of the carbohydrate content of biomass to fermentable monosaccharides by using glycoside hydrolases (GHs). Despite the vast number of GHs deposited in publicly available databases, 90 % of these enzymes are not characterized and many are unlikely to be suitable for industrial applications because of low activity and stability under processing conditions. While protein engineering (by using rational strategies as well as directed evolution) has been used to evolve GHs for industrial applications, bioprospecting and metagenomics provide alternative approaches to finding GHs with desired properties. The identification of new GHs from large genetic inventories relies on efficient protein expression and rapid high-throughput screening or selection to evaluate large enzyme libraries. However, heterologous expression of GHs (especially cellulases) in microbial hosts is usually very difficult, and often results in misfolded and/or inactive enzymes or non-native enzyme structures. Furthermore, the poor correlation between the activity of many GHs on soluble and insoluble substrates necessitates the highthroughput screening to be carried out on insoluble lignocellulosic substrates. Herein, we present the development of a one-pot high-throughput in vitro glycoside hydrolase (HIGH) expression and screening method by using insoluble lignocellulosic substrates. The HIGH platform can be adapted to screen for cellulases, xylanases, amylases, and b-glucosidases.
The attractiveness of fermentor cultures of Dioscorea deltoidea Wall. (Dioscoreaceae) as a source... more The attractiveness of fermentor cultures of Dioscorea deltoidea Wall. (Dioscoreaceae) as a source of diosgenin has been greatly improved by switching away from the traditional product recovery method that has been used in all previous studies. By using a known but little-used ...
Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge ... more Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge Tethya californiana was analyzed based on 16S rRNA gene sequences. One candidate division and nine bacterial phyla were detected, including members of all five subdivisions of Proteobacteria. Moreover, chloroplast-derived Stramenopiles- and Rhodophyta-affiliated 16S rRNA gene sequences were found and Stramenopiles represented the most abundant clones (30%) in the clone library. On the phylum-level, the microbial fingerprint of T. californiana showed a similar pattern as its Mediterranean relative T. aurantium. An interesting difference was that Cyanobacteria that were abundantly present in T. aurantium were not found in T. californiana, but that the latter sponges harbored phototrophic Stramenopiles instead. Surprisingly, the phototrophic microorganisms were evenly distributed over the inner and outer parts of the sponge tissue, which implies that they also reside in regions without dire...
Closely related to the "protein folding problem" is the issue of protein misfolding and... more Closely related to the "protein folding problem" is the issue of protein misfolding and aggregation. Protein aggregation has been associated with the pathologies of nearly 20 human diseases and presents serious difficulties during the manufacture of pharmaceutical proteins. Computational studies of multiprotein systems have recently emerged as a powerful complement to experimental efforts aimed at understanding the mechanisms of protein aggregation. We describe the thermodynamics of systems containing two lattice-model 64-mers. A parallel tempering algorithm abates problems associated with glassy systems and the weighted histogram analysis method improves statistical quality. The presence of a second chain has a substantial effect on single-chain conformational preferences. The melting temperature is substantially reduced, and the increase in the population of unfolded states is correlated with an increase in interactions between chains. The transition from two native chai...
Protein aggregation has been associated with a number of human diseases, and is a serious problem... more Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregatio...
Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the ex... more Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the experimental mixed-solute-exclusion method, MSE (introduced by Kuga), to obtain the solute-exclusion curve representing the amount of imbibed liquid inside the gel inaccessible for a solute of radius r. We use the Brownian motion model (developed by Cassasa) to convert the size-exclusion curve into the pore-size distribution, which gives the frequency of pore radius R as a function of R. This theoretically-based interpretation of MSE data leads to the Fredholm integral equation that we solve numerically. Results are reported for a series of hydrogels containing acrylamide and 3 % MAPTAC; the hydrogels differed in extent of cross-linking and/or initial concentration of monomer. Pore-size distributions shift to lower pore sizes with rising initial monomer concentration and with rising cross-linker-to-monomer ratio.
Background: Ionic liquid (IL) pretreatment could enable an economically viable route to produce b... more Background: Ionic liquid (IL) pretreatment could enable an economically viable route to produce biofuels by providing efficient means to extract sugars and lignin from lignocellulosic biomass. However, to realize this, novel IL-based processes need to be developed in order to minimize the overall production costs and accelerate commercial viability. In this study, two variants of IL-based processes are considered: one based on complete removal of the IL prior to hydrolysis using a water-wash (WW) step and the other based on a "one-pot" (OP) process that does not require IL removal prior to saccharification. Detailed techno-economic analysis (TEA) of these two routes was carried out to understand the cost drivers, economic potential (minimum ethanol selling price, MESP), and relative merits and challenges of each route. Results: At high biomass loading (50%), both routes exhibited comparable economic performance with an MESP of $6.3/gal. With the possible advances identified (reduced water or acid/base consumption, improved conversion in pretreatment, and lignin valorization), the MESP could be reduced to around $3/gal ($3.2 in the WW route and $2.8 in the OP route). Conclusions: It was found that, to be competitive at industrial scale, lowered cost of ILs used and higher biomass loadings (50%) are essential for both routes, and in particular for the OP route. Overall, while the economic potential of both routes appears to be comparable at higher biomass loadings, the OP route showed the benefit of lower water consumption at the plant level, an important cost and sustainability consideration for biorefineries.
Background: Trichoderma reesei is a key cellulase source for economically saccharifying cellulosi... more Background: Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60-70°C. Results: A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50-70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T m ) and improved half-life at 60°C (t 1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t 1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes. Conclusion: Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.
Clostridium acetobutylicum is a bacterial species that ferments sugar to a mixture of organic sol... more Clostridium acetobutylicum is a bacterial species that ferments sugar to a mixture of organic solvents (acetone, butanol and ethanol). This protocol delineates a methodology to combine solventogenic clostridial fermentation and chemical catalysis via extractive fermentation for the production of biofuel blendstocks. Extractive fermentation of C. acetobutylicum is operated in fed-batch mode with a concentrated feed solution (500 grams per liter glucose and 50 grams per liter yeast extract) for 60 h, producing in excess of 40 g of solvents (acetone, butanol and ethanol) between the completely immiscible extractant and aqueous phases of the bioreactor. After distillation of the extractant phase, the acetone, butanol and ethanol mixture is upgraded to long-chain ketones over a palladium-hydrotalcite (Pd-HT) catalyst. This reaction is generally carried out in batch with a high-pressure Q-tube for 20 h at 250 °C. Following this protocol enables the production of ∼0.5 g of high-value biofu...
A large number of novel compounds with significant medical potential have been isolated from spon... more A large number of novel compounds with significant medical potential have been isolated from sponges, motivating efforts to develop techniques for the sustainable cultivation of sponge biomass. To date, 33 sponges from nine different orders have been examined to assess their ability to be cultured in vitro. However, little consideration has been given to the relationships between these sponges; only one report has considering the phylogenetic relationships between the species. On the basis of morphological data, no taxonomic specificity was apparent as an indicator for the successful cultivation of the sponges. As the systematic classification of the Demospongiae is poorly understood, we collated available information on the success of in vitro sponge cell cultivation reports and examined the phylogenetic relationships of these sponges through the use of 18S and 28S rDNA sequence data. Based on molecular data, the ability of sponges to form primary aggregates from the dissociated cells of marine demosponges indicates that taxonomic trends may exist, emphasizing the need to better characterize sponges being investigated for biotechnological applications.
Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the ex... more Pore-size distributions have been measured for cationic acrylamide-based hydrogels. We use the experimental mixed-solute-exclusion method, MSE (introduced by Kuga), to obtain the solute-exclusion curve representing the amount of imbibed liquid inside the gel inaccessible for a solute of radius r. We use the Brownian motion model (developed by Cassasa) to convert the size-exclusion curve into the pore-size distribution, which gives the frequency of pore radius R as a function of R. This theoretically-based interpretation of MSE data leads to the Fredholm integral equation that we solve numerically. Results are reported for a series of hydrogels containing acrylamide and 3 % MAPTAC; the hydrogels differed in extent of cross-linking and/or initial concentration of monomer. Pore-size distributions shift to lower pore sizes with rising initial monomer concentration and with rising cross-linker-to-monomer ratio.
Sponges have rich and diverse associated microbial communities, which may have important function... more Sponges have rich and diverse associated microbial communities, which may have important functions in their metabolism. A survey of the archaeal communities of 23 poriferan species, focusing on the family Axinellidae, was conducted over the period 2002-2004 using 16S rDNA gene libraries created with archaeal-speciWc primers. The 28S rDNA sequences of the sponge hosts were also obtained. Of 23 species, 19 showed evidence of archaeal communities from group C1a (marine group I; Crenarchaeota), with three of these also showing evidence of Archaea from group E2 (marine group II; Euryarchaeota). Within the Crenarchaeota, two strongly supported sponge-speciWc clades were identiWed corresponding to the sponge family Axinellidae, and a novel sponge clade denoted clade C. These Wndings suggest that these archaea have evolved closely with their sponge hosts and are likely to play an important role in their metabolism.
Protein aggregation has been associated with a number of human diseases, and is a serious problem... more Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregation, although most association events take place at times much shorter than those required for folding. Last, we discuss results presented here as a basis for future work aimed at rational design of mutations to reduce aggregation propensity, as well as for development of small-molecular weight refolding enhancers. B 2004 Wiley Periodicals, Inc.
The addition of a biorefinery to an organic farm was investigated, where ethanol was produced fro... more The addition of a biorefinery to an organic farm was investigated, where ethanol was produced from germinated rye grains and whey, and the effluent was separated into two streams: the protein-rich solid fraction, to be used as animal feed, and the liquid fraction, which can be co-digested with clover grass silage to produce biogas. A method for ethanol production from rye was applied by utilizing inherent amylase activity from germination of the seed. Biogas potential of ethanol fermentation effluent was measured through anaerobic digestion trials. The effluent from the trials was assumed to serve as natural fertilizer. A technoeconomic analysis was also performed; total capital investment was estimated to be approximately 4 M USD. Setting a methane selling price according to available incentives for ''green electricity'' (0.72 USD/m 3 ) led to a minimum ethanol selling price of 1.89 USD/L (project lifetime 25 yr, at a discount rate 10%).
ABSTRACT This chapter contains sections titled: IntroductionProduction ProcessesBiomass Transport... more ABSTRACT This chapter contains sections titled: IntroductionProduction ProcessesBiomass Transportation and HandlingConversion of Biomass into SugarsConversion of Sugars into BiofuelsSeparation and PurificationCo-product HandlingMajor Cost DriversRisksPolicy SupportInfrastructure and Vehicle ModificationsConclusions Acknowledgments
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