Background:
Replacing the energy-intensive evaporation of stillage by anaerobic digestion is one way of decreasing the energy demand of the lignocellulosic biomass to the ethanol process. The biogas can be upgraded and sold as transportation fuel, injected directly into the gas grid or be incinerated on-site for combined heat and power generation. A techno-economic evaluation of the spruce-to-ethanol process, based on SO2-catalysed steam pretreatment followed by simultaneous saccharification and fermentation, has been performed using the commercial flow-sheeting program Aspen Plus™. Various process configurations of anaerobic digestion of the stillage, with different combinations of co-products, have been evaluated in terms of energy efficiency and ethanol production cost versus the reference case of evaporation.
Results:
Anaerobic digestion of the stillage showed a significantly higher overall energy efficiency (87-92%), based on the lower heating values, than the reference case (81%). Although the amount of ethanol produced was the same in all scenarios, the production cost varied between 4.00 and 5.27 Swedish kronor per litre (0.38-0.50 euro/L), including the reference case.
Conclusions:
Higher energy efficiency options did not necessarily result in lower ethanol production costs. Anaerobic digestion of the stillage with biogas upgrading was demonstrated to be a favourable option for both energy efficiency and ethanol production cost. The difference in the production cost of ethanol between using the whole stillage or only the liquid fraction in anaerobic digestion was negligible for the combination of co-products including upgraded biogas, electricity and district heat.

Background:
It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH) and endoglucanases (EG). Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble ?-cellulose ? considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto ?-cellulose.
Results:
First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir) and kinetics (pseudo-first-order). Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare ?-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured.It was found that, using ?-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto ?-cellulose and thus bolstered their activity. Complete suspension of ?-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble ?-cellulose.
Conclusions:
?-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation shows in detail, for the first time, the adsorption of purified cellulases onto ?-cellulose, the effect of hydrodynamics on cellulase adsorption and the correlation between the adsorption and the activity of cellulases at different hydrodynamic conditions. Complete suspension of the substrate has to be ensured in order to optimize the cellulase attack. In the future, screenings should be conducted with ?-cellulose so that proper cellulases are selected to best hydrolyze the real alkaline-pretreated biomass used in biorefineries.

Background:
Simultaneous saccharification and co-fermentation (SSCF) has been recognized as a feasible option for ethanol production from xylose-rich lignocellulosic materials. To reach high ethanol concentration in the broth, a high content of water-insoluble solids (WIS) is needed, which creates mixing problems and, furthermore, may decrease xylose uptake. Feeding of substrate has already been proven to give a higher xylose conversion than a batch SSCF. In the current work, enzyme feeding, in addition to substrate feeding, was investigated as a means of enabling a higher WIS content with a high xylose conversion in SSCF of a xylose-rich material. A recombinant xylose-fermenting strain of Saccharomyces cerevisiae (TMB3400) was used for this purpose in fed-batch SSCF experiments of steam-pretreated wheat straw.
Results:
By using both enzyme and substrate feeding, the xylose conversion in SSCF could be increased from 40% to 50% in comparison to substrate feeding only. In addition, by this design of the feeding strategy, it was possible to process a WIS content corresponding to 11% in SSCF and obtain an ethanol yield on fermentable sugars of 0.35 g g-1.
Conclusion:
A combination of enzyme and substrate feeding was shown to enhance xylose uptake by yeast and increase overall ethanol yield in SSCF. This is conceptually important for the design of novel SSCF processes aiming at high-ethanol titers. Substrate feeding prevents viscosity from becoming too high and thereby allows a higher total amount of WIS to be added in the process. The enzyme feeding, furthermore, enables keeping the glucose concentration low, which kinetically favors xylose uptake and results in a higher xylose conversion.

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Background:
To make lignocellulosic fuel ethanol economically competitive with fossil fuels, it is necessary to reduce the production cost. One way to achieve this is by increasing the substrate concentration in the production process, and thus reduce the energy demand in the final distillation of the fermentation broth. However, increased substrate concentration in simultaneous saccharification and fermentation (SSF) processes has been shown to result in reduced ethanol yields and severe stirring problems. Because the SSF medium is being continuously hydrolyzed, running the process in fed-batch mode could potentially reduce the stirring problems and lead to increased ethanol yields in high-solids SSF. Different enzyme feeding strategies, with the enzymes either present in the reactor from start-up or fed into the reactor together with the substrate, have been studied, along with the influence of the enzyme feeding strategy on the final ethanol yield and productivity.
Results:
In the present study, SSF was run successfully with 10% and 14% water-insoluble solids (WIS) in batch and fed-batch mode. The mixing of the material in the reactor was significantly better in fed-batch than batch mode, and similarly high or higher ethanol yields were achieved in fed-batch mode compared with batch SSF in some cases. No general trend in the dependence of ethanol yield on enzyme feeding strategy was found.
Conclusions:
The optimum enzyme feeding strategy appears to depend on the conditions during SSF, such as the WIS concentration and the concentration of inhibitory compounds in the SSF medium.

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.

Background:
The two-step dilute acid hydrolysis (DAH) of softwood is costly in energy demands and capital costs. However, it has the advantage that hydrolysis and subsequent removal of hemicellulose-derived sugars can be carried out under conditions of low severity, resulting in a reduction in the level of sugar degradation products during the more severe subsequent steps of cellulose hydrolysis. In this paper, we discuss a single-step DAH method that incorporates a temperature profile at two levels. This profile should simulate the two-step process while removing its major disadvantage, that is, the washing step between the runs, which leads to increased energy demand.
Results:
The experiments were conducted in a reactor with a controlled temperature profile. The total dry matter content of the hydrolysate was up to 21.1% w/w, corresponding to a content of 15.5% w/w of water insoluble solids. The highest measured glucose yield, (18.3 g glucose per 100 g dry raw material), was obtained after DAH cycles of 3 min at 209degreesC and 6 min at 211degreesC with 1% H2SO4, which resulted in a total of 26.3 g solubilized C6 sugars per 100g dry raw material. To estimate the remaining sugar potential, enzymatic hydrolysis (EH) of the solid fraction was also performed. EH of the solid residue increased the total level of solubilized C6 sugars to a maximum of 35.5 g per 100 g dry raw material when DAH was performed as described above (3 min at 210degreesC and 2 min at 211degreesC with 1% H2SO4).
Conclusion:
The dual-temperature DAH method did not yield decisively better results than the single-temperature, one-step DAH. When we compared the results with those of earlier studies, the hydrolysis performance was better than with the one-step DAH but not as well as that of the two-step, single-temperature DAH. Additional enzymatic hydrolysis resulted in lower levels of solubilized sugars compared with other studies on one-step DAH and two-step DAH followed by enzymatic hydrolysis. A two-step steam pretreatment with EH gave rise to a considerably higher sugar yield in this study.

Background:
Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced.
Results:
Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways.
Conclusion:
To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.

Background:
Corn grain is an important renewable source for bioethanol production in the USA. Corn ethanol is currently produced by steam liquefaction of starch-rich grains followed by enzymatic saccharification and fermentation. Corn stover (the non-grain parts of the plant) is a potential feedstock to produce cellulosic ethanol in second-generation biorefineries. At present, corn grain is harvested by removing the grain from the living plant while leaving the stover behind on the field. Alternatively, whole corn plants can be harvested to cohydrolyze both starch and cellulose after a suitable thermochemical pretreatment to produce fermentable monomeric sugars. In this study, we used physiologically immature corn silage (CS) and matured whole corn plants (WCP) as feedstocks to produce ethanol using ammonia fiber expansion (AFEX) pretreatment followed by enzymatic hydrolysis (at low enzyme loadings) and cofermentation (for both glucose and xylose) using a cellulase-amylase-based cocktail and a recombinant Saccharomyces cerevisiae 424A (LNH-ST) strain, respectively. The effect on hydrolysis yields of AFEX pretreatment conditions and a starch/cellulose-degrading enzyme addition sequence for both substrates was also studied.
Results:
AFEX-pretreated starch-rich substrates (for example, corn grain, soluble starch) had a 1.5-3-fold higher enzymatic hydrolysis yield compared with the untreated substrates. Sequential addition of cellulases after hydrolysis of starch within WCP resulted in 15-20% higher hydrolysis yield compared with simultaneous addition of hydrolytic enzymes. AFEX-pretreated CS gave 70% glucan conversion after 72 h of hydrolysis for 6% glucan loading (at 8 mg total enzyme loading per gram glucan). Microbial inoculation of CS before ensilation yielded a 10-15% lower glucose hydrolysis yield for the pretreated substrate, due to loss in starch content. Ethanol fermentation of AFEX-treated (at 6% w/w glucan loading) CS hydrolyzate (resulting in 28 g/L ethanol at 93% metabolic yield) and WCP (resulting in 30 g/L ethanol at 89% metabolic yield) is reported in this work.
Conclusions:
The current results indicate the feasibility of co-utilization of whole plants (that is, starchy grains plus cellulosic residues) using an ammonia-based (AFEX) pretreatment to increase bioethanol yield and reduce overall production cost.

Background:
Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.
Results:
The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation.All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.
Conclusions:
Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.

Background:
Grasses are relatively recalcitrant to genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L.) has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient gene expression in planta may lead to stable transformation methods with increased efficiency.
Results:
A high-throughput Agrobacterium-mediated transient gene expression system has been developed for in planta inoculation of germinating switchgrass seedlings. Four different Agrobacterium strains were compared for their ability to infect switchgrass seedlings, and strain AGL1 was found to be the most infective. Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of a reporter gene (GUSPlus, a variation of the beta-glucuronidase (GUS) gene). The addition of L-cysteine and dithiothreitol in the presence of acetosyringone significantly increased GUS expression compared with control treatments, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased GUS expression. 4-Methylumbelliferyl beta-D-galactopyranoside (MUG) assays showed a peak of beta-glucuronidase (GUS) enzyme activity 3 days after cocultivation with Agrobacterium harboring pCambia1305.2, whereas MUG assays showed a peak of enzyme activity 5 days after cocultivation with Agrobacterium harboring pCambia1305.1.
Conclusion:
Agrobacterium strains C58, GV3101 and EHA105 are less able to deliver transfer DNA to switchgrass seedlings (cultivar Alamo) compared with strain AGL1. Transient expression was increased by double or triple wounding treatments such as mixing by vortex with carborundum, sonication, separation by centrifugation, and heat shock. The addition of thiol compounds such as L-cysteine and dithiothreitol in combination with acetosyringone during cocultivation also increased transient expression. The combination of multiple wounding treatments along with the addition of thiol compounds during cocultivation increased transient expression levels from 6% to 54%. There were differences in temporal GUS expression induced by pCambia1305.1 and pCambia1305.2.

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.

Background:
US legislation requires the use of advanced biofuels to be made from non-food feedstocks. However, commercialization of lignocellulosic ethanol technology is more complex than expected and is therefore running behind schedule. This is creating a demand for non-food, but more easily converted, starch-based feedstocks other than corn that can fill the gap until the second generation technologies are commercially viable. Winter barley is such a feedstock but its mash has very high viscosity due to its high content of -glucans. This fact, along with a lower starch content than corn, makes ethanol production at the commercial scale a real challenge.
Results:
A new fermentation process for ethanol production from Thoroughbred, a winter barley variety with a high starch content, was developed. The new process was designated the EDGE (enhanced dry grind enzymatic) process. In this process, in addition to the normal starch-converting enzymes, two accessory enzymes were used to solve the beta-glucan problem. First, beta-glucanases were used to hydrolyze the beta-glucans to oligomeric fractions, thus significantly reducing the viscosity to allow good mixing for the distribution of the yeast and nutrients. Next, beta-glucosidase was used to complete the beta-glucan hydrolysis and to generate glucose, which was subsequently fermented in order to produce additional ethanol. While beta-glucanases have been previously used to improve barley ethanol production by lowering viscosity, this is the first full report on the benefits of adding beta-glucosidases to increase the ethanol yield.
Conclusions:
In the EDGE process, 30% of total dry solids could be used to produce 15% v/v ethanol. Under optimum conditions an ethanol yield of 402 L/MT (dry basis) or 2.17 gallons/53 lb bushel of barley with 15% moisture was achieved. The distillers dried grains with solubles (DDGS) co-product had extremely low beta-glucan (below 0.2%) making it suitable for use in both ruminant and mono-gastric animal feeds.