http://www.biotechnologyforbiofuels.com The most accessed research articles published by Biotechnology for Biofuels 2012-05-06T00:00:00Z Background: A major challenge in the identification and development of superior feedstocks for the production of second generation biofuels is the rapid assessment of biomass composition in a large number of samples. Currently, highly accurate and precise robotic analysis systems are available for the evaluation of biomass composition, on a large number of samples, with a variety of pretreatments. However, the lack of an inexpensive and high-throughput process for large scale sampling of biomass resources is still an important limiting factor. Our goal was to develop a simple mechanical maize stalk core sampling device that can be utilized to collect uniform samples of a dimension compatible with robotic processing and analysis, while allowing the collection of hundreds to thousands of samples per day. Results: We have developed a core sampling device (CSD) to collect maize stalk samples compatible with robotic processing and analysis. The CSD facilitates the collection of thousands of uniform tissue cores consistent with high-throughput analysis required for breeding, genetics, and production studies. With a single CSD operated by one person with minimal training, more than 1,000 biomass samples were obtained in an eight-hour period. One of the main advantages of using cores is the high level of homogeneity of the samples obtained and the minimal opportunity for sample contamination. In addition, the samples obtained with the CSD can be placed directly into a bath of ice, dry ice, or liquid nitrogen maintaining the composition of the biomass sample for relatively long periods of time. Conclusions: The CSD has been demonstrated to successfully produce homogeneous stalk core samples in a repeatable manner with a throughput substantially superior to the currently available sampling methods. Given the variety of maize developmental stages and the diversity of stalk diameter evaluated, it is expected that the CSD will have utility for other bioenergy crops as well. http://www.biotechnologyforbiofuels.com/content/5/1/27 German Muttoni James Johnson Nicholas Santoro Craig Rhiner Karl Haro von Mogel Shawn Kaeppler Natalia de Leon Biotechnology for Biofuels 2012, null:27 2012-05-01T00:00:00Z doi:10.1186/1754-6834-5-27 /content/figures/1754-6834-5-27-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 27 2012-05-01T00:00:00Z PDF Background: Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-beta-1,4-glucanases, cellobiohydrolases, cellodextrinases, and beta-glucosidases. Converting cellobiose by beta-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results: In this study, a cDNA encoding beta-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40oC and pH 5~6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50oC by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5L was added to the SSF system. Conclusion: Our characterizations of the novel beta-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40oC. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.Keywordsendoglucanase, beta-glucosidase, Neocallimastix patriciarum, rumen fungi, simultaneous saccharification and fermentation. http://www.biotechnologyforbiofuels.com/content/5/1/24 Hsin-Liang Chen Yo-Chia Chen Mei-Yeh Lu Jui-Jen Chang Hiaow-Ting Wang Tzi-Yuan Wang Sz-Kai Ruan Tao-Yuan Wang Kuo-Yen Hung Hsing-Yi Cho Huei-Mien Ke Wan-Ting Lin Ming-Che Shih Wen-Hsiung Li Biotechnology for Biofuels 2012, null:24 2012-04-19T00:00:00Z doi:10.1186/1754-6834-5-24 /content/figures/1754-6834-5-24-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 24 2012-04-19T00:00:00Z PDF Background: Bioethanol produced from the lignocellulosic fractions of sugar cane (bagasse and leaves), i.e. second generation (2G) bioethanol, has a promising market potential as an automotive fuel; however, the process is still under investigation on pilot/demonstration scale. From a process perspective, improvements in plant design can lower the production cost, providing better profitability and competitiveness if the conversion of the whole sugar cane is considered. Simulations have been performed with AspenPlus to investigate how process integration can affect the minimum ethanol selling price of this 2G process (MESP-2G), as well as improve the plant energy efficiency. This is achieved by integrating the well-established sucrose-to-bioethanol process with the enzymatic process for lignocellulosic materials. Bagasse and leaves were steam pretreated using H3PO4 as catalyst and separately hydrolysed and fermented. Results: The addition of a steam dryer, doubling of the enzyme dosage in enzymatic hydrolysis, including leaves as raw material in the 2G process, heat integration and the use of more energy-efficient equipment led to a 37 % reduction in MESP-2G compared to the Base case. Modelling showed that the MESP for 2G ethanol was 0.97 US$/L, while in the future it could be reduced to 0.78 US$/L. In this case the overall production cost of 1G + 2G ethanol would be about 0.40 US$/L with an output of 102 L/ton dry sugar cane including 50 % leaves. Sensitivity analysis of the future scenario showed that a 50 % decrease in the cost of enzymes, electricity or leaves would lower the MESP-2G by about 20%, 10% and 4.5%, respectively. Conclusions: According to the simulations, the production of 2G bioethanol from sugar cane bagasse and leaves in Brazil is already competitive (without subsidies) with 1G starch-based bioethanol production in Europe. Moreover 2G bioethanol could be produced at a lower cost if subsidies were used to compensate for the opportunity cost from the sale of excess electricity and if the cost of enzymes continues to fall. http://www.biotechnologyforbiofuels.com/content/5/1/22 Stefano Macrelli Johan Mogensen Guido Zacchi Biotechnology for Biofuels 2012, null:22 2012-04-13T00:00:00Z doi:10.1186/1754-6834-5-22 /content/figures/1754-6834-5-22-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 22 2012-04-13T00:00:00Z XML This paper reviews recent literature on bioenergy potentials in conjunction with available biomass conversion technologies. The geographical scope is the European Union, which has set a course for long term development of its energy supply from the current dependence on fossil resources to a dominance of renewable resources. A cornerstone in European energy policies and strategies is biomass and bioenergy. The annual demand for biomass for energy is estimated to increase from the current level of 5.7 EJ to 10.0 EJ in 2020. Assessments of bioenergy potentials vary substantially due to methodological inconsistency and assumptions applied by individual authors. Forest biomass, agricultural residues and energy crops constitute the three major sources of biomass for energy, with the latter probably developing into the most important over the 21st century. Land use and the changes thereof is a key issue in sustainable bioenergy production as land availability is an ultimately limiting factor. http://www.biotechnologyforbiofuels.com/content/5/1/25 Niclas Bentsen Claus Felby Biotechnology for Biofuels 2012, null:25 2012-04-30T00:00:00Z doi:10.1186/1754-6834-5-25 /content/figures/1754-6834-5-25-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 25 2012-04-30T00:00:00Z PDF Background: The recent discovery of accessory proteins that boost cellulose hydrolysis has increased the economical and technical efficiency of processing cellulose to bioethanol. Oxidative enzymes (e.g. GH61) present in new commercial enzyme preparations have shown to increase cellulose conversion yields. When using pure cellulose substrates it has been determined that both oxidized and unoxidized cellodextrin products are formed. We report the effect of oxidative activity in a commercial enzyme mix (Cellic CTec2) upon overall hydrolysis, formation of oxidized products and impact on beta-glucosidase activity. The experiments were done at high solids loadings using a lignocellulosic substrate simulating commercially relevant conditions. Results: The Cellic CTec2 contained oxidative enzymes which produce gluconic acid from lignocellulose. Both gluconic and cellobionic acid were produced during hydrolysis of pretreated wheat straw at 30% WIS. Up to 4% of released glucose was oxidized into gluconic acid using Cellic CTec2, whereas no oxidized products were detected when using an earlier cellulase preparation Celluclast/Novozym188. However, the cellulose conversion yield was 25% lower using Celluclast/Novozym188 compared to Cellic CTec2. Despite the advantage of the oxidative enzymes, it was shown that aldonic acids could be problematic to the hydrolytic enzymes. Hydrolysis experiments revealed that cellobionic acid was hydrolyzed by beta-glucosidase at a rate almost 10-fold lower than for cellobiose, and the formed gluconic acid was an inhibitor of the beta-glucosidase.Interestingly, the level of gluconic acid varied significantly with temperature. At 50C (SHF conditions) 35% less gluconic acid was produced compared to at 33C (SSF conditions). We also found that in the presence of lignin, no reducing agent is needed for the function of the oxidative enzymes. Conclusions: The presence of oxidative enzymes in Cellic CTec2 led to the formation of cellobionic and gluconic acid during hydrolysis of pretreated wheat straw and filter paper. Gluconic acid was a stronger inhibitor of beta-glucosidase than glucose. The formation of oxidized products decreased as the hydrolysis temperature was increased from 33C to 50C. Despite end-product inhibition, the oxidative cleavage of the cellulose chains has a synergistic effect upon the overall hydrolysis of cellulose as the sugar yield increased compared to using an enzyme preparation without oxidative activity. http://www.biotechnologyforbiofuels.com/content/5/1/26 David Cannella Chia-wen Hsieh Claus Felby Henning Jørgensen Biotechnology for Biofuels 2012, null:26 2012-04-30T00:00:00Z doi:10.1186/1754-6834-5-26 /content/figures/1754-6834-5-26-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 26 2012-04-30T00:00:00Z PDF Biodiesel is a promising alternative, and renewable, fuel. As its production increases, so does production of the principle co-product, crude glycerol. The effective utilization of crude glycerol will contribute to the viability of biodiesel. In this review, composition and quality factors of crude glycerol are discussed. The value-added utilization opportunities of crude glycerol are reviewed. The majority of crude glycerol is used as feedstock for production of other value-added chemicals, followed by animal feeds. http://www.biotechnologyforbiofuels.com/content/5/1/13 Fangxia Yang Milford Hanna Runcang Sun Biotechnology for Biofuels 2012, null:13 2012-03-14T00:00:00Z doi:10.1186/1754-6834-5-13 /content/figures/1754-6834-5-13-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 13 2012-03-14T00:00:00Z XML Background: Cost-effective production of lignocellulosic biofuels remains a major financial and technical challenge at the industrial scale. A critical tool in biofuels process development is the techno-economic (TE) model, which calculates biofuel production costs using a process model and an economic model. The process model solves mass and energy balances for each unit, and the economic model estimates capital and operating costs from the process model based on economic assumptions. The process model inputs include experimental data on the feedstock composition and intermediate product yields for each unit. These experimental yield data are calculated from primary measurements. Uncertainty in these primary measurements is propagated to the calculated yields, to the process model, and ultimately to the economic model. Thus, outputs of the TE model have a minimum uncertainty associated with the uncertainty in the primary measurements. Results: We calculate the uncertainty in the Minimum Ethanol Selling Price (MESP) estimate for lignocellulosic ethanol production via a biochemical conversion process: dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis and co-fermentation of the resulting sugars to ethanol. We perform a sensitivity analysis on the TE model and identify the feedstock composition and conversion yields from three unit operations (xylose from pretreatment, glucose from enzymatic hydrolysis, and ethanol from fermentation) as the most important variables. The uncertainty in the pretreatment xylose yield arises from multiple measurements, whereas the glucose and ethanol yields from enzymatic hydrolysis and fermentation, respectively, are dominated by a single measurement: the fraction of insoluble solids (fIS) in the biomass slurries. Conclusions: We calculate a $0.15/gal uncertainty in MESP from the TE model due to uncertainties in primary measurements. This result sets a lower bound on the error bars of the TE model predictions. This analysis highlights the primary measurements that merit further development to reduce the uncertainty associated with their use in TE models. While we develop and apply this mathematical framework to a specific biorefinery scenario here, this analysis can be readily adapted to other types of biorefining processes and provides a general framework for propagating uncertainty due to analytical measurements through a TE model. http://www.biotechnologyforbiofuels.com/content/5/1/23 Kristin Vicari Sai Sandeep Tallam Tatyana Shatova Kang Joo Koh Christopher Scarlata David Humbird Edward Wolfrum Gregg Beckham Biotechnology for Biofuels 2012, null:23 2012-04-17T00:00:00Z doi:10.1186/1754-6834-5-23 /content/figures/1754-6834-5-23-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 23 2012-04-17T00:00:00Z PDF Background: Understanding the dynamics of the microbial communities that, along with their secreted enzymes, are involved in the natural process of biomass composting may hold the key to breaking the major bottleneck in biomass-to-biofuels conversion technology, which is the still-costly deconstruction of polymeric biomass carbohydrates to fermentable sugars. However, the complexity of both the structure of plant biomass and its counterpart microbial degradation communities makes it difficult to investigate the composting process. Results: In this study, a composter was set up with a mix of yellow poplar (Liriodendron tulipifera) wood-chips and mown lawn grass clippings (85:15 in dry-weight) and used as a model system. The microbial rDNA abundance data obtained from analyzing weekly-withdrawn composted samples suggested population-shifts from bacteria-dominated to fungus-dominated communities. Further analyses by an array of optical microscopic, transcriptional and enzyme-activity techniques yielded correlated results, suggesting that such population shifts occurred along with early removal of hemicellulose followed by attack on the consequently uncovered cellulose as the composting progressed. Conclusion: The observed shifts in dominance by representative microbial groups, along with the observed different patterns in the gene expression and enzymatic activities between cellulases, hemicellulases, and ligninases during the composting process, provide new perspectives for biomass-derived biotechnology such as consolidated bioprocessing (CBP) and solid-state fermentation for the production of cellulolytic enzymes and biofuels. http://www.biotechnologyforbiofuels.com/content/5/1/20 Hui Wei Melvin Tucker John Baker Michelle Harris Yonghua Luo Qi Xu Michael Himmel Shi-You Ding Biotechnology for Biofuels 2012, null:20 2012-04-10T00:00:00Z doi:10.1186/1754-6834-5-20 /content/figures/1754-6834-5-20-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 20 2012-04-10T00:00:00Z PDF Although measurements of crystallinity index (CI) have a long history, it has been found that CI varies significantly depending on the choice of measurement method. In this study, four different techniques incorporating X-ray diffraction and solid-state 13C nuclear magnetic resonance (NMR) were compared using eight different cellulose preparations. We found that the simplest method, which is also the most widely used, and which involves measurement of just two heights in the X-ray diffractogram, produced significantly higher crystallinity values than did the other methods. Data in the literature for the cellulose preparation used (Avicel PH-101) support this observation. We believe that the alternative X-ray diffraction (XRD) and NMR methods presented here, which consider the contributions from amorphous and crystalline cellulose to the entire XRD and NMR spectra, provide a more accurate measure of the crystallinity of cellulose. Although celluloses having a high amorphous content are usually more easily digested by enzymes, it is unclear, based on studies published in the literature, whether CI actually provides a clear indication of the digestibility of a cellulose sample. Cellulose accessibility should be affected by crystallinity, but is also likely to be affected by several other parameters, such as lignin/hemicellulose contents and distribution, porosity, and particle size. Given the methodological dependency of cellulose CI values and the complex nature of cellulase interactions with amorphous and crystalline celluloses, we caution against trying to correlate relatively small changes in CI with changes in cellulose digestibility. In addition, the prediction of cellulase performance based on low levels of cellulose conversion may not include sufficient digestion of the crystalline component to be meaningful. http://www.biotechnologyforbiofuels.com/content/3/1/10 Sunkyu Park John Baker Michael Himmel Philip Parilla David Johnson Biotechnology for Biofuels 2010, null:10 2010-05-24T00:00:00Z doi:10.1186/1754-6834-3-10 /content/figures/1754-6834-3-10-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 10 2010-05-24T00:00:00Z XML Background: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. Results: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than the similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. Conclusion: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium. http://www.biotechnologyforbiofuels.com/content/5/1/30 Adam Guss Daniel Olson Nicky Caiazza Lee Lynd Biotechnology for Biofuels 2012, null:30 2012-05-06T00:00:00Z doi:10.1186/1754-6834-5-30 /content/figures/1754-6834-5-30-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 30 2012-05-06T00:00:00Z PDF