http://www.biotechnologyforbiofuels.com The latest research articles published by Biotechnology for Biofuels 2012-01-19T00:00:00Z Background: Microbial lipids have drawn increasing attention in recent years as promising raw materials for biodiesel production, and the use of lignocellulosic hydrolysates as carbon sources seems to be a feasible strategy for cost-effective lipid fermentation with oleaginous microorganisms on a large scale. During the hydrolysis of lignocellulosic materials with dilute acid, however, various kinds of inhibitors, especially large amounts of organic acids, will be produced, which substantially decrease the fermentability of lignocellulosic hydrolysates. To overcome the inhibitory effects of organic acids, it is critical to understand their impact on the growth and lipid accumulation of oleaginous microorganisms. Results: In our present work, we investigated for the first time the effect of ten representative organic acids in lignocellulosic hydrolysates on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans cells. In contrast to previous reports, we found that the toxicity of the organic acids to the cells was not directly related to their hydrophobicity. It is worth noting that most organic acids tested were less toxic than aldehydes to the cells, and some could even stimulate the growth and lipid accumulation at a low concentration. Unlike aldehydes, most binary combinations of organic acids exerted no synergistic inhibitory effects on lipid production. The presence of organic acids decelerated the consumption of glucose, whereas it influenced the utilization of xylose in a different and complicated way. In addition, all the organic acids tested, except furoic acid, inhibited the malic activity of T. fermentans. Furthermore, the inhibition of organic acids on cell growth was dependent more on inoculum size, temperature and initial pH than on lipid content. Conclusions: This work provides some meaningful information about the effect of organic acid in lignocellulosic hydrolysates on the lipid production of oleaginous yeast, which is helpful for optimization of biomass hydrolysis processes, detoxified pretreatment of hydrolysates and lipid production using lignocellulosic materials. http://www.biotechnologyforbiofuels.com/content/5/1/4 Chao Huang Hong Wu Zong-jun Liu Jun Cai Wen-yong Lou Min-hua Zong Biotechnology for Biofuels 2012, null:4 2012-01-19T00:00:00Z doi:10.1186/1754-6834-5-4 /content/figures/1754-6834-5-4-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 4 2012-01-19T00:00:00Z PDF 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 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 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 Here is described a method based on BODIPY staining, fluorescence activated cell sorting (FACS) and microplate-based isolation of lipid-rich microalgae from an environmental sample. Our results show that direct sorting onto solid medium upon FACS can save about three weeks during the scale-up process as compared with the growth of the same cultures in liquid medium. This approach enabled us to isolate a biodiverse collection of several axenic and unialgal cultures of different phyla. http://www.biotechnologyforbiofuels.com/content/4/1/61 Hugo Pereira Luisa Barreira Andre Mozes Claudia Florindo Cristina Polo Catarina Duarte Luisa Custodio Joao Varela Biotechnology for Biofuels 2011, null:61 2011-12-22T00:00:00Z doi:10.1186/1754-6834-4-61 /content/figures/1754-6834-4-61-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 61 2011-12-22T00:00:00Z PDF Background: Due to the complexity of lignocellulosic materials, a complete enzymatic hydrolysis into fermentable sugars requires a variety of cellulolytic and xylanolytic enzymes. Addition of xylanases has been shown to significantly improve the performance of cellulases and to increase cellulose hydrolysis by solubilizing xylans in lignocellulosic materials. The goal of this work was to investigate the effect of acetyl xylan esterase (AXE) originating from Trichoderma reesei on xylan solubilization and enzymatic hydrolysis of cellulose. Results: The solubilization of xylan in pretreated wheat straw and giant reed (Arundo donax) by xylanolytic enzymes and the impact of the sequential or simultaneous solubilization of xylan on the hydrolysis of cellulose by purified enzymes were investigated. The results showed that the removal of acetyl groups in xylan by AXE increased the accessibility of xylan to xylanase and improved the hydrolysis of xylan in pretreated wheat straw and giant reed. Solubilization of xylan led to an increased accessibility of cellulose to cellulases and thereby increased the hydrolysis extent of cellulose. A clear synergistic effect between cellulases and xylanolytic enzymes was observed. The highest hydrolysis yield of cellulose was obtained with a simultaneous use of cellulases, xylanase and AXE, indicating the presence of acetylated xylan within the cellulose matrix. Acetylated xylobiose and acetylated xylotriose were produced from xylan without AXE, as confirmed by atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry. Conclusions: The results in this paper demonstrate that supplementation of xylanase with AXE enhances the solubilization of xylan to some extent and, consequently, increases the subsequent hydrolysis of cellulose. The highest hydrolysis yield was, however, obtained by simultaneous hydrolysis of xylan and cellulose, indicating a layered structure of cellulose and xylan chains in the cell wall substrate. AXE has an important role in the hydrolysis of lignocellulosic materials containing acetylated xylan. http://www.biotechnologyforbiofuels.com/content/4/1/60 Junhua Zhang Matti Siika-aho Maija Tenkanen Liisa Viikari Biotechnology for Biofuels 2011, null:60 2011-12-20T00:00:00Z doi:10.1186/1754-6834-4-60 /content/figures/1754-6834-4-60-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 60 2011-12-20T00:00:00Z XML Background: Contamination of bacteria in large scale yeast fermentations is a serious problem and threat to the development of successful biofuel production plants. Huge research efforts have been spent in order to solve this problem, but additional ways must still be found to keep bacterial contaminants from thriving in these environments. The aim of this project was to develop process conditions that would inhibit bacterial growth while giving yeast a competitive advantage. Results: Lactic acid bacteria are usually considered to be the most common contaminants in industrial yeast fermentations. Our observations support this view but also suggest that acetic acid bacteria, although not so numerous, could be a much more problematic obstacle to overcome. Acetic acid bacteria showed a capacity to drastically reduce the viability of yeast. In addition, they consumed the previously formed ethanol. Lactic acid bacteria did not show this detrimental effect on yeast viability. It was possible to combat both types of bacteria by a combined addition of NaCl and ethanol to the wood hydrolysate medium used. As a result of NaCl + ethanol additions the amount of viable bacteria decreased and yeast viability was enhanced concomitantly with an increase in ethanol concentration. The successful result obtained via addition of NaCl and ethanol was also confirmed in a real industrial ethanol production plant with its natural inherent yeast/bacterial community. Conclusions: It is possible to reduce the number of bacteria and offer a selective advantage to yeast by a combined addition of NaCl and ethanol when cultivated in lignocellulosic medium such as wood hydrolysate. However, for optimal results, the concentrations of NaCl + ethanol must be adjusted to suit the challenges offered by each hydrolysate. http://www.biotechnologyforbiofuels.com/content/4/1/59 Eva Albers Emma Johansson Carl-Johan Franzen Christer Larsson Biotechnology for Biofuels 2011, null:59 2011-12-20T00:00:00Z doi:10.1186/1754-6834-4-59 /content/figures/1754-6834-4-59-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 59 2011-12-20T00:00:00Z PDF Background: Large-scale production of effective cellulose hydrolytic enzymes is the key to bioconversion of agricultural residues to ethanol. The goal of this study was to develop rice plant as a bioreactor for large-scale production of cellulose hydrolytic enzymes via genetic transformation and to improve rice straw simultaneously as an efficient biomass feedstock for conversion of cellulose to glucose. Results: In this study, the cellulose hydrolytic enzyme beta-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained, which expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analysis demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39oC and another hour at 81oC yielded 43% more reducing sugars than wild type rice straw. Conclusion: Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in transgenic rice may also facilitate saccharification of cellulose in rice straw and significantly reduce the costs for hydrolytic enzymes. http://www.biotechnologyforbiofuels.com/content/4/1/58 Hong-Li Chou Ziyu Dai Chia-Wen Hsieh Maurice Ku Biotechnology for Biofuels 2011, null:58 2011-12-10T00:00:00Z doi:10.1186/1754-6834-4-58 /content/figures/1754-6834-4-58-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 58 2011-12-10T00:00:00Z PDF Background: The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during Very High Gravity (VHG) or lignocellulosic fermentations. In this study, a set of Saccharomyces cerevisiae genes found to confer resistance to the simultaneous presence of different relevant stresses, through genome-wide screenings, were identified as required for maximal fermentation performance under industrial conditions. Results: Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of BUD31 and HPR1 was found to lead to the increase of both ethanol yield and fermentation rate, while PHO85, VRP1 and YGL024w expression is required for maximal ethanol production in VHG fermentations. Five genes, ERG2, PRS3, RAV1, RPB4 and VMA8 were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or for maximal fermentation rate of this substrate. Conclusions: The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers. http://www.biotechnologyforbiofuels.com/content/4/1/57 Francisco Pereira Pedro Guimaraes Daniel Gomes Nuno Mira Miguel Teixeira Isabel Sa-Correia Lucilia Domingues Biotechnology for Biofuels 2011, null:57 2011-12-09T00:00:00Z doi:10.1186/1754-6834-4-57 /content/figures/1754-6834-4-57-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 57 2011-12-09T00:00:00Z PDF Background: Solid acid catalyst was prepared from Kraft lignin by chemical activation with phosphoric acid, pyrolysis and sulfuric acid. This catalyst had high acid density as characterized by scanning electron microscope (SEM), energy-dispersive x-ray spectrometry (EDX), and Brunauer, Emmett and Teller (BET) method analyses. It was further used to catalyze the esterification of oleic acid and one-step conversion of non-pretreated Jatropha oil to biodiesel. The effects of catalyst loading, reaction temperature and oil-to-methanol molar ratio, on the catalytic activity of the esterification were investigated. Results: The highest catalytic activity was achieved with a 96.1% esterification rate, and the catalyst can be reused three times with little deactivation under optimized conditions. Biodiesel production from Jatropha oil was studied under such conditions. It was found that 96.3% biodiesel yield from non-pretreated Jatropha oil with high-acid value (12.7 mg KOH/g) could be achieved. Conclusions: The catalyst can be easily separated for reuse. This single-step process could be a potential route for biodiesel production from high-acid value oil by simplifying the procedure and reducing costs. http://www.biotechnologyforbiofuels.com/content/4/1/56 Fei-ling Pua Zhen Fang Sarani Zakaria Feng Guo Chin-hua Chia Biotechnology for Biofuels 2011, null:56 2011-12-07T00:00:00Z doi:10.1186/1754-6834-4-56 /content/figures/1754-6834-4-56-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 56 2011-12-07T00:00:00Z PDF