http://www.biotechnologyforbiofuels.com The most accessed research articles published by Biotechnology for Biofuels 2012-01-13T00:00:00Z Background: The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. Results: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. Conclusions: The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass. http://www.biotechnologyforbiofuels.com/content/5/1/2 Yongchao Li Timothy Tschaplinski Nancy Engle Choo Hamilton Miguel Rodriguez James Liao Christopher Schadt Adam Guss Yunfeng Yang David Graham Biotechnology for Biofuels 2012, null:2 2012-01-04T00:00:00Z doi:10.1186/1754-6834-5-2 /content/figures/1754-6834-5-2-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 2 2012-01-04T00:00:00Z XML A major challenge when using microorganisms to produce bulk chemicals such as biofuels is that the production targets are often toxic to cells. Many biofuels are known to reduce cell viability through damage to the cell membrane and interference with essential physiological processes. Therefore, cells must trade off biofuel production and survival, reducing potential yields. Recently, there have been several efforts towards engineering strains for biofuel tolerance. Promising methods include engineering biofuel export systems, heat shock proteins, membrane modifications, more general stress responses, and approaches that integrate multiple tolerance strategies. In addition, in situ recovery methods and media supplements can help to ease the burden of end-product toxicity and may be used in combination with genetic approaches. Recent advances in systems and synthetic biology provide a framework for tolerance engineering. This review highlights recent targeted approaches towards improving microbial tolerance to next-generation biofuels with a particular emphasis on strategies that will improve production. http://www.biotechnologyforbiofuels.com/content/4/1/32 Mary Dunlop Biotechnology for Biofuels 2011, null:32 2011-09-21T00:00:00Z doi:10.1186/1754-6834-4-32 /content/figures/1754-6834-4-32-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 32 2011-09-21T00:00:00Z XML Background: Improving the hydrolytic performance of hemicellulases on lignocellulosic biomass is of considerable importance for second generation biorefining. To address this problem, and also to gain greater understanding of structure-function relationships, especially related to xylanase action on complex biomass, we have implemented a combinatorial strategy to engineer the GH11 xylanase (Tx-Xyn) from Thermobacillus xylanilyticus. Results: Following in vitro enzyme evolution and screening on wheat straw, nine best-performing clones were identified, which display mutations at positions 3, 6, 27 and 111. All of these mutants showed increased hydrolytic activity on wheat straw, and solubilised arabinoxylans that were not modified by the parental enzyme. Compared to the wild type enzyme, the most active mutants, S27T and Y111T, increased the solubilisation of arabinoxylans from depleted wheat straw 2.3-fold and 2.1-fold respectively. In addition, five mutants, S27T, Y111H, Y111S, Y111T and S27T-Y111H increased total hemicellulose conversion of intact wheat straw from 16.7%tot. xyl (wild-type Tx-Xyn) to 18.6 - 20.4%tot. xyl. Also, all five mutant enzymes exhibited a better ability to act in synergy with a cellulase cocktail (Accellerase 1500), thus procuring increases in overall wheat straw hydrolysis. Conclusions: Analysis of the results allows us to hypothesize that the increased hydrolytic ability of the mutants is linked to i) improved ligand binding in a putative secondary binding site, ii) the diminution of surface hydrophobicity, and/or iii) the modification of thumb flexibility, induced by mutations at position 111. Nevertheless, the relatively modest improvements that were observed also underline the fact that enzyme engineering alone cannot overcome the limits imposed by the complex organisation of the plant cell wall and the lignin barrier. http://www.biotechnologyforbiofuels.com/content/5/1/3 Letian Song Beatrice Siguier Claire Dumon Sophie Bozonnet Michael O'Donohue Biotechnology for Biofuels 2012, null:3 2012-01-13T00:00:00Z doi:10.1186/1754-6834-5-3 /content/figures/1754-6834-5-3-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 3 2012-01-13T00:00:00Z 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: The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies. Results: Aiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414. Conclusions: Using this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production. http://www.biotechnologyforbiofuels.com/content/5/1/1 Andre Schuster Kenneth Bruno James Collett Scott Baker Bernhard Seiboth Christian Kubicek Monika Schmoll Biotechnology for Biofuels 2012, null:1 2012-01-02T00:00:00Z doi:10.1186/1754-6834-5-1 /content/figures/1754-6834-5-1-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 1 2012-01-02T00:00:00Z XML Background: Transesterification of Jatropha oil was carried out in t-butanol solvent using immobilized lipase from Enterobacter aerogenes. The presence of t-butanol significantly reduced the negative effects caused by both methanol and glycerol. The effects of various reaction parameters on transesterification of Jatropha oil were studied. Results: The maximum yield of biodiesel was 94% (of which 68% conversion was achieved with respect to methyl oleate) with an oil:methanol molar ratio of 1:4, 50 U of immobilized lipase/g of oil, and a t-butanol:oil volume ratio of 0.8:1 at 55°C after 48 h of reaction time. There was negligible loss in lipase activity even after repeated use for seven cycles. Conclusion: To the best of our knowledge this is the first report on biodiesel synthesis using immobilized E. aerogenes lipase. http://www.biotechnologyforbiofuels.com/content/2/1/1 Annapurna Kumari Paramita Mahapatra Vijay Kumar Garlapati Rintu Banerjee Biotechnology for Biofuels 2009, null:1 2009-01-14T00:00:00Z doi:10.1186/1754-6834-2-1 /content/figures/1754-6834-2-1-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 1 2009-01-14T00:00:00Z XML Background: In recent years, biorefining of lignocellulosic biomass to produce multi-products such as ethanol and other biomaterials has become a dynamic research area. Pretreatment technologies that fractionate sugarcane bagasse are essential for the successful use of this feedstock in ethanol production. In this paper, we investigate modifications in the morphology and chemical composition of sugarcane bagasse submitted to a two-step treatment, using diluted acid followed by a delignification process with increasing sodium hydroxide concentrations. Detailed chemical and morphological characterization of the samples after each pretreatment condition, studied by high performance liquid chromatography, solid-state nuclear magnetic resonance, diffuse reflectance Fourier transformed infrared spectroscopy and scanning electron microscopy, is reported, together with sample crystallinity and enzymatic digestibility. Results: Chemical composition analysis performed on samples obtained after different pretreatment conditions showed that up to 96% and 85% of hemicellulose and lignin fractions, respectively, were removed by this two-step method when sodium hydroxide concentrations of 1% (m/v) or higher were used. The efficient lignin removal resulted in an enhanced hydrolysis yield reaching values around 100%. Considering the cellulose loss due to the pretreatment (maximum of 30%, depending on the process), the total cellulose conversion increases significantly from 22.0% (value for the untreated bagasse) to 72.4%. The delignification process, with consequent increase in the cellulose to lignin ratio, is also clearly observed by nuclear magnetic resonance and diffuse reflectance Fourier transformed infrared spectroscopy experiments. We also demonstrated that the morphological changes contributing to this remarkable improvement occur as a consequence of lignin removal from the sample. Bagasse unstructuring is favored by the loss of cohesion between neighboring cell walls, as well as by changes in the inner cell wall structure, such as damaging, hole formation and loss of mechanical resistance, facilitating liquid and enzyme access to crystalline cellulose. Conclusions: The results presented herewith show the efficiency of the proposed method for improving the enzymatic digestibility of sugarcane bagasse and provide understanding of the pretreatment action mechanism. Combining the different techniques applied in this work warranted thorough information about the undergoing morphological and chemical changes and was an efficient approach to understand the morphological effects resulting from sample delignification and its influence on the enhanced hydrolysis results. http://www.biotechnologyforbiofuels.com/content/4/1/54 Camila Rezende Marisa Lima Priscila Maziero Eduardo deAzevedo Wanius Garcia Igor Polikarpov Biotechnology for Biofuels 2011, null:54 2011-11-28T00:00:00Z doi:10.1186/1754-6834-4-54 /content/figures/1754-6834-4-54-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 54 2011-11-28T00:00:00Z XML Background: While advantages of biofuel have been widely reported, studies also highlight the challenges in large scale production of biofuel. Cost of ethanol and process energy use in cellulosic ethanol plants are dependent on technologies used for conversion of feedstock. Process modeling can aid in identifying techno-economic bottlenecks in a production process. A comprehensive techno-economic analysis was performed for conversion of cellulosic feedstock to ethanol using some of the common pretreatment technologies: dilute acid, dilute alkali, hot water and steam explosion. Detailed process models incorporating feedstock handling, pretreatment, simultaneous saccharification and co-fermentation, ethanol recovery and downstream processing were developed using SuperPro Designer. Tall Fescue (Festuca arundinacea Schreb) was used as a model feedstock. Results: Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/dry metric ton biomass for conversion process using dilute acid, dilute alkali, hot water and steam explosion pretreatment technologies respectively. Price of feedstock and cellulose enzymes were assumed as $50/metric ton and 0.517/kg broth (10% protein in broth, 600 FPU/g protein) respectively. Capital cost of ethanol plants processing 250,000 metric tons of feedstock/year was $1.92, $1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanol was estimated for production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Water use in the production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment was estimated 5.96, 6.07, 5.84 and 4.36 kg/L ethanol respectively. Conclusions: Ethanol price and energy use were highly dependent on process conditions used in the ethanol production plant. Potential for significant ethanol cost reductions exist in increasing pentose fermentation efficiency and reducing biomass and enzyme costs. The results demonstrated the importance of addressing the tradeoffs in capital costs, pretreatment and downstream processing technologies. http://www.biotechnologyforbiofuels.com/content/4/1/27 Deepak Kumar Ganti Murthy Biotechnology for Biofuels 2011, null:27 2011-09-05T00:00:00Z doi:10.1186/1754-6834-4-27 /content/figures/1754-6834-4-27-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 27 2011-09-05T00:00:00Z XML Background: Bioethanol can be produced from sugar-rich, starch-rich (first generation; 1G) or lignocellulosic (second generation; 2G) raw materials. Integration of 2G ethanol with 1G could facilitate the introduction of the 2G technology. The capital cost per ton of fuel produced would be diminished and better utilization of the biomass can be achieved. It would, furthermore, decrease the energy demand of 2G ethanol production and also provide both 1G and 2G plants with heat and electricity. In the current study, steam-pretreated wheat straw (SPWS) was mixed with presaccharified wheat meal (PWM) and converted to ethanol in simultaneous saccharification and fermentation (SSF). Results: Both the ethanol concentration and the ethanol yield increased with increasing amounts of PWM in mixtures with SPWS. The maximum ethanol yield (99% of the theoretical yield, based on the available C6 sugars) was obtained with a mixture of SPWS containing 2.5% water-insoluble solids (WIS) and PWM containing 2.5% WIS, resulting in an ethanol concentration of 56.5 g/L. This yield was higher than those obtained with SSF of either SPWS (68%) or PWM alone (91%). Conclusions: Mixing wheat straw with wheat meal would be beneficial for both 1G and 2G ethanol production. However, increasing the proportion of WIS as wheat straw and the possibility of consuming the xylose fraction with a pentose-fermenting yeast should be further investigated. http://www.biotechnologyforbiofuels.com/content/3/1/16 Borbala Erdei Zsolt Barta Balint Sipos Kati Reczey Mats Galbe Guido Zacchi Biotechnology for Biofuels 2010, null:16 2010-07-02T00:00:00Z doi:10.1186/1754-6834-3-16 /content/figures/1754-6834-3-16-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 16 2010-07-02T00:00:00Z XML Background: Two economic factors make watermelon worthy of consideration as a feedstock for ethanol biofuel production. First, about 20% of each annual watermelon crop is left in the field because of surface blemishes or because they are misshapen; currently these are lost to growers as a source of revenue. Second, the neutraceutical value of lycopene and L-citrulline obtained from watermelon is at a threshold whereby watermelon could serve as starting material to extract and manufacture these products. Processing of watermelons to produce lycopene and L-citrulline, yields a waste stream of watermelon juice at the rate of over 500 L/t of watermelons. Since watermelon juice contains 7 to 10% (w/v) directly fermentable sugars and 15 to 35 μmol/ml of free amino acids, its potential as feedstock, diluent, and nitrogen supplement was investigated in fermentations to produce bioethanol. Results: Complete watermelon juice and that which did not contain the chromoplasts (lycopene), but did contain free amino acids, were readily fermentable as the sole feedstock or as diluent, feedstock supplement, and nitrogen supplement to granulated sugar or molasses. A minimum level of ~400 mg N/L (~15 μmol/ml amino nitrogen) in watermelon juice was required to achieve maximal fermentation rates when it was employed as the sole nitrogen source for the fermentation. Fermentation at pH 5 produced the highest rate of fermentation for the yeast system that was employed. Utilizing watermelon juice as diluent, supplemental feedstock, and nitrogen source for fermentation of processed sugar or molasses allowed complete fermentation of up to 25% (w/v) sugar concentration at pH 3 (0.41 to 0.46 g ethanol per g sugar) or up to 35% (w/v) sugar concentration at pH 5 with a conversion to 0.36 to 0.41 g ethanol per g sugar. Conclusion: Although watermelon juice would have to be concentrated 2.5- to 3-fold to serve as the sole feedstock for ethanol biofuel production, the results of this investigation indicate that watermelon juice, either as whole juice fermented on-site or as a waste stream from neutraceutical production, could easily integrate with other more concentrated feedstocks where it could serve as diluent, supplemental feedstock, and nitrogen supplement. http://www.biotechnologyforbiofuels.com/content/2/1/18 Wayne Fish Benny Bruton Vincent Russo Biotechnology for Biofuels 2009, null:18 2009-08-26T00:00:00Z doi:10.1186/1754-6834-2-18 Watermelon juice as a biofuel Concentrated watermelon juice, extracted from unsaleable fruit can be used directly or as a diluent for ethanol production. /content/figures/1754-6834-2-18-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 18 2009-08-26T00:00:00Z XML