A Novel Kinetic Modeling of Enzymatic Hydrolysis of Sugarcane Bagasse Pretreated by Hydrothermal and Organosolv Processes.

Lignocellulosic biomasses have a complex and compact structure, requiring physical and/or chemical pretreatments to produce glucose before hydrolysis. Mathematical modeling of enzymatic hydrolysis highlights the interactions between cellulases and cellulose, evaluating the factors contributing to reactor scale-up and conversion rates. Furthermore, this study evaluated the influence of two pretreatments (hydrothermal and organosolv) on the kinetics of enzymatic hydrolysis of sugarcane bagasse. The kinetic parameters of the model were estimated using the Pikaia genetic algorithm with data from the experimental profiles of cellulose, cellobiose, glucose, and xylose. The model considered the phenomenon of non-productive adsorption of cellulase on lignin and inhibition of cellulase by xylose. Moreover, it included the behavior of cellulase adsorption on the substrate throughout hydrolysis and kinetic equations for obtaining xylose from xylanase-catalyzed hydrolysis of xylan. The model for both pretreatments was experimentally validated with bagasse concentration at 10% w/v. The Plackett-Burman design identified 17 kinetic parameters as significant in the behavior of process variables. In this way, the modeling and parameter estimation methodology obtained a good fit from the experimental data and a more comprehensive model.

Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production.

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

A Novel Fungal Lipase With Methanol Tolerance and Preference for Macaw Palm Oil.

Macaw palm is a highly oil-producing plant, which presents high contents of free fatty acids, being a promising feedstock for biofuel production. The current chemical routes are costly and complex, involving highly harsh industrial conditions. Enzymatic processing is a potential alternative; however, it is hampered by the scarce knowledge on biocatalysts adapted to this acidic feedstock. This work describes a novel lipase isolated from the thermophilic fungus Rasamsonia emersonii (ReLip), which tolerates extreme conditions such as the presence of methanol, high temperatures, and acidic medium. Among the tested feedstocks, the enzyme showed the highest preference for macaw palm oil, producing a hydrolyzate with a final free fatty acid content of 92%. Crystallographic studies revealed a closed conformation of the helical amphipathic lid that typically undergoes conformational changes in a mechanism of interfacial activation. Such conformation of the lid is stabilized by a salt bridge, not observed in other structurally characterized homologs, which is likely involved in the tolerance to organic solvents. Moreover, the lack of conservation of the aromatic cluster IxxWxxxxxF in the lid of ReLip with the natural mutation of the phenylalanine by an alanine might be correlated with the preference of short acyl chains, although preserving catalytic activity on insoluble substrates. In addition, the presence of five acidic amino acids in the lid of ReLip, a rare property reported in other lipases, may have contributed to its ability to tolerate and be effective in acidic environments. Therefore, our work describes a new fungal biocatalyst capable of efficiently hydrolyzing macaw oil, an attractive feedstock for the production of "drop-in" biofuels, with high desirable feature for industrial conditions such as thermal and methanol tolerance, and optimum acidic pH. Moreover, the crystallographic structure was elucidated, providing a structural basis for the enzyme substrate preference and tolerance to organic solvents.

Techno-economic analysis and climate change impacts of sugarcane biorefineries considering different time horizons.

BACKGROUND: Ethanol production from lignocellulosic feedstocks (also known as 2nd generation or 2G ethanol process) presents a great potential for reducing both ethanol production costs and climate change impacts since agricultural residues and dedicated energy crops are used as feedstock. This study aimed at the quantification of the economic and environmental impacts considering the current and future scenarios of sugarcane biorefineries taking into account not only the improvements of the industrial process but also of biomass production systems. Technology assumptions and scenarios setup were supported by main companies and stakeholders, involved in the lignocellulosic ethanol production chain from Brazil and abroad. For instance, scenarios considered higher efficiencies and lower residence times for pretreatment, enzymatic hydrolysis, and fermentation (including pentoses fermentation); higher sugarcane yields; and introduction of energy cane (a high fiber variety of cane). RESULTS: Ethanol production costs were estimated for different time horizons. In the short term, 2G ethanol presents higher costs compared to 1st generation (1G) ethanol. However, in the long term, 2G ethanol is more competitive, presenting remarkable lower production cost than 1G ethanol, even considering some uncertainties regarding technology and market aspects. In addition, environmental assessment showed that both 1G (in the medium and long term) and 2G ethanol can reduce climate change impacts by more than 80% when compared to gasoline. CONCLUSIONS: This work showed the great potential of 2G ethanol production in terms of economic and environmental aspects. These results can support new research programs and public policies designed to stimulate both production and consumption of 2G ethanol in Brazil, accelerating the path along the learning curve. Some examples of mechanisms include: incentives to the establishment of local equipment and enzyme suppliers; and specific funding programs for the development and use of energy cane.

Investigation of uncertainties associated with the production of n-butanol through ethanol catalysis in sugarcane biorefineries.

This study evaluated the viability of n-butanol production integrated within a first and second generation sugarcane biorefinery. The evaluation included a deterministic analysis as well as a stochastic approach, the latter using Monte Carlo simulation. Results were promising for n-butanol production in terms of revenues per tonne of processed sugarcane, but discouraging with respect to internal rate of return (IRR). The uncertainty analysis determined there was high risk involved in producing n-butanol and co-products from ethanol catalysis. It is unlikely that these products and associated production route will be financially attractive in the short term without lower investment costs, supportive public policies and tax incentives coupled with biofuels' production strategies.

Adsorption characteristics of cellulase and ß-glucosidase on Avicel, pretreated sugarcane bagasse, and lignin.

Although adsorption is an essential step in the enzymatic hydrolysis of lignocellulosic materials, literature reports controversial results in relation to the adsorption of the cellulolitic enzymes on different biomasses/pretreatments, which makes difficult the description of this phenomenon in hydrolysis mathematical models. In this work, the adsorption of these enzymes on Avicel and sugarcane bagasse pretreated by the hydrothermal bagasse (HB) and organosolv bagasse (OB) methods was evaluated. The results have shown no significant adsorption of ß-glucosidase on Avicel or HB. Increasing solids concentration from 5% (w/v) to 10% (w/v) had no impact on the adsorption of cellulase on the different biomasses if stirring rates were high enough (>100 rpm for Avicel and >150 rpm for HB and OB). Adsorption equilibrium time was low for Avicel (10 Min) when compared with the lignocellulosic materials (120 Min). Adsorption isotherms determined at 4 and 50 °C have shown that for Avicel there was a decrease in the maximum adsorption capacity (Emax) with the temperature increase, whereas for HB increasing temperature increased Emax . Also, Emax increased with the content of lignin in the material. Adsorption studies of cellulase on lignin left after enzymatic digestion of HB show lower but significant adsorption capacity (Emax = 11.92 ± 0.76 mg/g).

Utilization of pentoses from sugarcane biomass: techno-economics of biogas vs. butanol production.

This paper presents the techno-economics of greenfield projects of an integrated first and second-generation sugarcane biorefinery in which pentose sugars obtained from sugarcane biomass are used either for biogas (consumed internally in the power boiler) or n-butanol production via the ABE batch fermentation process. The complete sugarcane biorefinery was simulated using Aspen Plus®. Although the pentoses stream available in the sugarcane biorefinery gives room for a relatively small biobutanol plant (7.1-12 thousand tonnes per year), the introduction of butanol and acetone to the product portfolio of the biorefinery increased and diversified its revenues. Whereas the IRR of the investment on a biorefinery with biogas production is 11.3%, IRR varied between 13.1% and 15.2% in the butanol production option, depending on technology (regular or engineered microorganism with improved butanol yield and pentoses conversion) and target market (chemicals or automotive fuels). Additional discussions include the effects of energy-efficient technologies for butanol processing on the profitability of the biorefinery.

Butanol production in a first-generation Brazilian sugarcane biorefinery: technical aspects and economics of greenfield projects.

The techno-economics of greenfield projects of a first-generation sugarcane biorefinery aimed to produce ethanol, sugar, power, and n-butanol was conducted taking into account different butanol fermentation technologies (regular microorganism and mutant strain with improved butanol yield) and market scenarios (chemicals and automotive fuel). The complete sugarcane biorefinery with the batch acetone-butanol-ethanol (ABE) fermentation process was simulated using Aspen Plus®. The biorefinery was designed to process 2 million tonne sugarcane per year and utilize 25%, 50%, and 25% of the available sugarcane juice to produce sugar, ethanol, and butanol, respectively. The investment on a biorefinery with butanol production showed to be more attractive [14.8% IRR, P(IRR>12%)=0.99] than the conventional 50:50 (ethanol:sugar) annexed plant [13.3% IRR, P(IRR>12%)=0.80] only in the case butanol is produced by an improved microorganism and traded as a chemical.

Integrated versus stand-alone second generation ethanol production from sugarcane bagasse and trash.

Ethanol production from lignocellulosic materials is often conceived considering independent, stand-alone production plants; in the Brazilian scenario, where part of the potential feedstock (sugarcane bagasse) for second generation ethanol production is already available at conventional first generation production plants, an integrated first and second generation production process seems to be the most obvious option. In this study stand-alone second generation ethanol production from surplus sugarcane bagasse and trash is compared with conventional first generation ethanol production from sugarcane and with integrated first and second generation; simulations were developed to represent the different technological scenarios, which provided data for economic and environmental analysis. Results show that the integrated first and second generation ethanol production process from sugarcane leads to better economic results when compared with the stand-alone plant, especially when advanced hydrolysis technologies and pentoses fermentation are included.

Second generation ethanol in Brazil: can it compete with electricity production?

Much of the controversy surrounding second generation ethanol production arises from the assumed competition with first generation ethanol production; however, in Brazil, where bioethanol is produced from sugarcane, sugarcane bagasse and trash will be used as feedstock for second generation ethanol production. Thus, second generation ethanol production may be primarily in competition with electricity production from the lignocellulosic fraction of sugarcane. A preliminary technical and economic analysis of the integrated production of first and second generation ethanol from sugarcane in Brazil is presented and different technological scenarios are evaluated. The analysis showed the importance of the integrated use of sugarcane including the biomass represented by surplus bagasse and trash that can be taken from the field. Second generation ethanol may favorably compete with bioelectricity production when sugarcane trash is used and when low cost enzyme and improved technologies become commercially available.

Simulation of integrated first and second generation bioethanol production from sugarcane: comparison between different biomass pretreatment methods.

Sugarcane bagasse is used as a fuel in conventional bioethanol production, providing heat and power for the plant; therefore, the amount of surplus bagasse available for use as raw material for second generation bioethanol production is related to the energy consumption of the bioethanol production process. Pentoses and lignin, byproducts of the second generation bioethanol production process, may be used as fuels, increasing the amount of surplus bagasse. In this work, simulations of the integrated bioethanol production process from sugarcane, surplus bagasse and trash were carried out. Selected pre-treatment methods followed, or not, by a delignification step were evaluated. The amount of lignocellulosic materials available for hydrolysis in each configuration was calculated assuming that 50% of sugarcane trash is recovered from the field. An economic risk analysis was carried out; the best results for the integrated first and second generation ethanol production process were obtained for steam explosion pretreatment, high solids loading for hydrolysis and 24-48 h hydrolysis. The second generation ethanol production process must be improved (e.g., decreasing required investment, improving yields and developing pentose fermentation to ethanol) in order for the integrated process to be more economically competitive.

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha.

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.