http://www.biotechnologyforbiofuels.com The latest research articles published by Biotechnology for Biofuels 2013-05-17T00:00:00Z Background: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. Results: The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold beta-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high beta-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars. Conclusions: The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail. http://www.biotechnologyforbiofuels.com/content/6/1/79 Etienne Jourdier Céline Cohen Laurent Poughon Christian Larroche Frédéric Monot Fadhel Ben Chaabane Biotechnology for Biofuels 2013, null:79 2013-05-17T00:00:00Z doi:10.1186/1754-6834-6-79 /content/figures/1754-6834-6-79-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 79 2013-05-17T00:00:00Z PDF Background: The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris. Results: Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either beta-d-xylosidase (EC 3.2.1.37) or alpha-l-arabinofuranosidase (EC 3.2.1.55) activity, while others displayed the ability to degrade AZCL-xylan or AZCL-beta-(1,3)-beta-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens. Conclusions: This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study. http://www.biotechnologyforbiofuels.com/content/6/1/78 Géraldine Bastien Grégory Arnal Sophie Bozonnet Sandrine Laguerre Fernando Ferreira Régis Fauré Bernard Henrissat Fabrice Lefèvre Patrick Robe Olivier Bouchez Céline Noirot Claire Dumon Michael O¿Donohue Biotechnology for Biofuels 2013, null:78 2013-05-14T00:00:00Z doi:10.1186/1754-6834-6-78 /content/figures/1754-6834-6-78-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 78 2013-05-14T00:00:00Z PDF Switchgrass (Panicum virgatum L.) is a C4 perennial warm season grass indigenous to the North American tallgrass prairie. A number of its natural and agronomic traits, including adaptation to a wide geographical distribution, low nutrient requirements and production costs, high water use efficiency, high biomass potential, ease of harvesting, and potential for carbon storage, make it an attractive dedicated biomass crop for biofuel production. We believe that genetic improvements using biotechnology will be important to realize the potential of the biomass and biofuel-related uses of switchgrass. Tissue culture techniques aimed at rapid propagation of switchgrass and genetic transformation protocols have been developed. Rapid progress in genome sequencing and bioinformatics has provided efficient strategies to identify, tag, clone and manipulate many economically-important genes, including those related to higher biomass, saccharification efficiency, and lignin biosynthesis. Application of the best genetic tools should render improved switchgrass that will be more economically and environmentally sustainable as a lignocellulosic bioenergy feedstock. http://www.biotechnologyforbiofuels.com/content/6/1/77 Madhugiri Nageswara-Rao Jaya Soneji Charles Kwit C Stewart Biotechnology for Biofuels 2013, null:77 2013-05-12T00:00:00Z doi:10.1186/1754-6834-6-77 /content/figures/1754-6834-6-77-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 77 2013-05-12T00:00:00Z PDF Background: The C4 perennial grass Miscanthusgiganteus has proved to be a promising bio-energy crop. However, the biomass recalcitrance is a major challenge in biofuel production. Effective pretreatment is necessary for achieving a high efficiency in converting the crop to fermentable sugars, and subsequently biofuels and other valued products. Results: Miscanthus lutarioriparious was pretreated with a liquid hot water (LHW) reactor. Between the pretreatment severity (PS) of 2.56-4.71, the solid recovery was reduced; cellulose recovery remained nearly unchanged; and the Klason lignin content was slightly increased which was mainly due to the dissolving of hemicellulose and the production of a small amount of pseudo-lignin. The result shows that a LHW PS of 4.71 could completely degrade the hemicellulose in Miscanthus. Hemicellulose removal dislodged the enzymatic barrier of cellulose, and the ethanol conversion of 98.27% was obtained. Conclusions: Our study demonstrated that LHW served as an effective pretreatment in case that Miscanthus lutarioriparious was used for ethanol production by simultaneous saccharification and fermentation. The combination and the pretreatment method and Miscanthus feedstock hold a great potential for biofuel production. http://www.biotechnologyforbiofuels.com/content/6/1/76 Hong-Qiang Li Cheng-Lan Li Tao Sang Jian Xu Biotechnology for Biofuels 2013, null:76 2013-05-10T00:00:00Z doi:10.1186/1754-6834-6-76 /content/figures/1754-6834-6-76-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 76 2013-05-10T00:00:00Z PDF Background: In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results: Compositional analysis revealed an increase in the cellulose content, reaching around 81c and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion: Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue. http://www.biotechnologyforbiofuels.com/content/6/1/75 Marisa Lima Gabriela Lavorente Hana da Silva Juliano Bragatto Camila Rezende Oigres Bernardinelli Eduardo de Azevedo Leonardo Gomez Simon McQueen-Mason Carlos Labate Igor Polikarpov Biotechnology for Biofuels 2013, null:75 2013-05-09T00:00:00Z doi:10.1186/1754-6834-6-75 /content/figures/1754-6834-6-75-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 75 2013-05-09T00:00:00Z PDF Background: Succinic acid is one of the key platform chemicals which can be produced via biotechnology process instead of petrochemical process. Biomass derived bio-oil have been investigated intensively as an alternative of diesel and gasoline fuels. Bio-oil could be fractionized into organic phase and aqueous phase parts. The organic phase bio-oil can be easily upgraded to transport fuel. The aqueous phase bio-oil (AP-bio-oil) is of low value. There is no report for its usage or upgrading via biological methods. In this paper, the use of AP-bio-oil for the production of succinic acid was investigated. Results: The transgenic E. coli strain could grow in modified M9 medium containing 20 v/v% AP-bio-oil with an increase in OD from 0.25 to 1.09. And 0.38 g/L succinic acid was produced. With the presence of 4 g/L glucose in the medium, succinic acid concentration increased from 1.4 to 2.4 g/L by addition of 20 v/v% AP-bio-oil. When enzymatic hydrolysate of corn stover was used as carbon source, 10.3 g/L succinic acid was produced. The obtained succinic acid concentration increased to 11.5 g/L when 12.5 v/v% AP-bio-oil was added. However, it decreased to 8 g/L when 50 v/v% AP-bio-oil was added. GC-MS analysis revealed that some low molecular carbon compounds in the AP-bio-oil were utilized by E. coli. Conclusions: The results indicate that AP-bio-oil can be used by E. coli for cell growth and succinic acid production. http://www.biotechnologyforbiofuels.com/content/6/1/74 Caixia Wang Anders Thygesen Yilan Liu Qiang Li Maohua Yang Dan Dang Ze Wang Yinhua Wan Weigang Lin Jianmin Xing Biotechnology for Biofuels 2013, null:74 2013-05-08T00:00:00Z doi:10.1186/1754-6834-6-74 /content/figures/1754-6834-6-74-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 74 2013-05-08T00:00:00Z XML Background: Many bacteria efficiently degrade lignocellulose yet the underpinning genome-wide metabolic and regulatory networks remain elusive. Here we revealed the “cellulose degradome” for the model mesophilic cellulolytic bacterium Clostridium cellulolyticum ATCC 35319, via an integrated analysis of its complete genome, its transcriptomes under glucose, xylose, cellobiose, cellulose, xylan or corn stover and its extracellular proteomes under glucose, cellobiose or cellulose. Results: Proteins for core metabolic functions, environment sensing, gene regulation and polysaccharide metabolism were enriched in the cellulose degradome. Analysis of differentially expressed genes revealed a “core” set of 48 CAZymes required for degrading cellulose-containing substrates as well as an “accessory” set of 76 CAZymes required for specific non-cellulose substrates. Gene co-expression analysis suggested that Carbon Catabolite Repression (CCR) related regulators sense intracellular glycolytic intermediates and control the core CAZymes that mainly include cellulosomal components, whereas 11 sets of Two-Component Systems (TCSs) respond to availability of extracellular soluble sugars and respectively regulate most of the accessory CAZymes and associated transporters. Surprisingly, under glucose alone, the core cellulases were highly expressed at both transcript and protein levels. Furthermore, glucose enhanced cellulolysis in a dose-dependent manner, via inducing cellulase transcription at low concentrations. Conclusion: A molecular model of cellulose degradome in C. cellulolyticum (Ccel) was proposed, which revealed the substrate-specificity of CAZymes and the transcriptional regulation of core cellulases by CCR where the glucose acts as a CCR inhibitor instead of a trigger. These features represent a distinct environment-sensing strategy for competing while collaborating for cellulose utilization, which can be exploited for process and genetic engineering of microbial cellulolysis. http://www.biotechnologyforbiofuels.com/content/6/1/73 Chenggang Xu Ranran Huang Lin Teng Dongmei Wang Christopher Hemme Ilya Borovok Qiang He Raphael Lamed Edward Bayer Jizhong Zhou Jian Xu Biotechnology for Biofuels 2013, null:73 2013-05-08T00:00:00Z doi:10.1186/1754-6834-6-73 /content/figures/1754-6834-6-73-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 73 2013-05-08T00:00:00Z XML Background: Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed. Results: This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples. Conclusion: Our study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulated the global metabolic status, redirects metabolic flux of fixed CO2 into starch synthesis branch resulting in starch accumulation in L. punctata. http://www.biotechnologyforbiofuels.com/content/6/1/72 Xiang Tao Yang Fang Yao Xiao Yan-ling Jin Xin-rong Ma Yun Zhao Kai-ze He Hai Zhao Hai-yan Wang Biotechnology for Biofuels 2013, null:72 2013-05-08T00:00:00Z doi:10.1186/1754-6834-6-72 /content/figures/1754-6834-6-72-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 72 2013-05-08T00:00:00Z XML Background: Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, “absent technological breakthroughs”, it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold. Results: We have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance. Conclusions: Our results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production. http://www.biotechnologyforbiofuels.com/content/6/1/71 Hui Shen Charleson Poovaiah Angela Ziebell Timothy Tschaplinski Sivakumar Pattathil Erica Gjersing Nancy Engle Rui Katahira Yunqiao Pu Robert Sykes Fang Chen Arthur Ragauskas Jonathan Mielenz Michael Hahn Mark Davis C Stewart Richard Dixon Biotechnology for Biofuels 2013, null:71 2013-05-07T00:00:00Z doi:10.1186/1754-6834-6-71 /content/figures/1754-6834-6-71-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 71 2013-05-07T00:00:00Z XML Background: Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed trasesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially. Results: We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50[degree sign]C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis. Conclusions: Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties. http://www.biotechnologyforbiofuels.com/content/6/1/70 Tyler Korman Bobby Sahachartsiri David Charbonneau Grace Huang Marc Beauregard James Bowie Biotechnology for Biofuels 2013, null:70 2013-05-07T00:00:00Z doi:10.1186/1754-6834-6-70 /content/figures/1754-6834-6-70-toc.gif Biotechnology for Biofuels 1754-6834 ${item.volume} 70 2013-05-07T00:00:00Z PDF