The potential of cellulosic ethanol production from grasses in Thailand.

The grasses in Thailand were analyzed for the potentiality as the alternative energy crops for cellulosic ethanol production by biological process. The average percentage composition of cellulose, hemicellulose, and lignin in the samples of 18 types of grasses from various provinces was determined as 31.85-38.51, 31.13-42.61, and 3.10-5.64, respectively. The samples were initially pretreated with alkaline peroxide followed by enzymatic hydrolysis to investigate the enzymatic saccharification. The total reducing sugars in most grasses ranging from 500-600 mg/g grasses (70-80% yield) were obtained. Subsequently, 11 types of grasses were selected as feedstocks for the ethanol production by simultaneous saccharification and cofermentation (SSCF). The enzymes, cellulase and xylanase, were utilized for hydrolysis and the yeasts, Saccharomyces cerevisiae and Pichia stipitis, were applied for cofermentation at 35 °C for 7 days. From the results, the highest yield of ethanol, 1.14 g/L or 0.14 g/g substrate equivalent to 32.72% of the theoretical values was obtained from Sri Lanka ecotype vetiver grass. When the yields of dry matter were included in the calculations, Sri Lanka ecotype vetiver grass gave the yield of ethanol at 1,091.84 L/ha/year, whereas the leaves of dwarf napier grass showed the maximum yield of 2,720.55 L/ha/year (0.98 g/L or 0.12 g/g substrate equivalent to 30.60% of the theoretical values).

Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris.

The glucan 1,3-beta-glucosidase A gene (exgA) from Aspergillus oryzae and fused to the Saccharomyces cerevisiae signal peptide (α-factor) was expressed under the control of either a constitutive (GAP) or an inducible (AOX1) promoter in Pichia pastoris. A 1.4-fold higher extracellular enzyme activity (2 U/ml) was obtained using the AOX1 inducible expression system than with the GAP constitutive promoter (1.4 U/ml). The purified recombinant ExgA enzyme, with a yield of 10 mg protein/l culture supernatant, was about 40 kDa by SDS-PAGE analysis with a specific activity of 289 U/mg protein. The enzyme was optimally active at 35 °C and pH 5.0 and displayed a K(M) and V(max) of 0.56 mM and 10,042 µmol/(min mg protein), respectively, with p-nitrophenyl-ß-D-glucopyranoside as the substrate. Moreover, it was tolerant to glucose inhibition with a K(i) of 365 mM.

Butanol and ethanol production from tapioca starch wastewater by Clostridium spp.

Total (TWW) and tapioca starch wash wastewater (TSWW) from a cassava processing plant in Thailand were analyzed for their composition with a view to evaluate their potential as substrates for solvent production by ABE fermentation with Clostridium spp. Starch was detected at a 1.63-fold higher level in the TWW than that in the TSWW (24.4% and 15.0% (w/w), respectively). The chemical oxygen demand (COD) was broadly similar (20,093 and 20,433 mg/L), but the biological oxygen demand (BOD) was 1.84-fold higher (18,000 and 9,750 mg/L) in the TWW than that in the TSWW. Thus, the TSWW was selected as a substrate to evaluate its potential for butanol and ethanol production by two Clostridium spp. The combined ethanol plus butanol production in the TSWW at pH 6.5 was higher than that at pH 4.5, being around 27.8- and 3.4-fold higher in C. butyricum TISTR 1032 and C. acetobutylicum ATCC 824, respectively. In both strains, the butanol (and combined butanol plus ethanol) production level was improved at pH 5.5. The addition of yeast extract increased the bacterial cell production, but did not significantly improve solvent productivity in C. acetobutylicum, and even decreased butanol production by C. butyricum.

Effect of beta-cyclodextrin in artificial chaperones assisted foam fractionation of cellulase.

Foam fractionation has the potential to be a low-cost protein separation process; however, it may cause protein denaturation during the foaming process. In previous work with cellulase, artificial chaperones were integrated into the foam fractionation process in order to reduce the loss of enzymatic activity. In this study, other factors were introduced to further reduce the loss of cellulase activity: type of cyclodextrin, cyclodextrin concentration, dilution ratio cyclodextrin to the foamate and holding time. alpha-Cyclodextrin was almost as effective as beta-cyclodextrin in refolding the foamed cellulase-Cetyltrimethylammonium bromide mixture. beta-Cyclodextrin (6.5 mM) was almost as effective as 13 mM beta-cyclodextrin in refolding. The dilution ratio, seven parts foamate and three parts beta-cyclodextrin solution, was found to be most effective among the three ratios tested (7:3, 1:1, and 3:7). The activity after refolding at this dilution ratio is around 0.14 unit/mL. The refolding time study showed that the refolding process was found to be most effective for the short refolding times (within 1 h).

Enhancing cellulase foam fractionation with addition of surfactant.

Foam fractionation cannot be used to recover cellulase from an aerated water solution effectively because cellulase by itself can produce only a small amount of foam. The addition of a surfactant can, however, increase the foamate volume and enhance the concentration of cellulase. We studied three detergents individually added to a 200 mg/L cellulase solution to promote foaming. These detergents were anionic, cationic, and nonionic surfactants, respectively. Although contributing to foam production, it was observed that nonionic surfactant (Pluronic F-68) barely concentrated cellulase, leaving the enrichment ratio unchanged, near 1. With anionic surfactant, sodium dedecyl sulfate, and cationic surfactant, cetyltrimethylammonium bromide (CTAB), the enrichment ratio became much larger, but cellulase denaturation occurred, reducing the activity of the enzyme. When CTAB was used to help foam cellulase, beta-cyclodextrin was subsequently added to the foamate to help restore the enzyme activity.

The effect of pH on the foam fractionation of beta-glucosidase and cellulase.

The surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.

Degeneration of beta-glucosidase activity in a foam fractionation process.

Foam fractionation is a promising technique for concentrating proteins because of its simplicity and low operating cost. One such protein that can be foamed is the enzyme cellulase. The use of inexpensively purified cellulase may be a key step in the economical production of ethanol from biomass. We conducted foam fractionation experiments at total reflux using the cellulase component beta-glucosidase to study how continuous shear affects beta-glucosidase in a foam such as a fermentation or foam fractionation process. The experiments were conducted at pH 2.4, 5.4, and 11.6 and airflow rates of 3, 6, 15, 20, and 32 cc/min to determine how beta-glucosidase activity changes in time at these different conditions. This is apparently a novel and simple way of testing for changes in enzyme activity within a protein foam. The activity did not degenerate during 5 min of reflux at pH 5.4 at an airflow rate of 10 cc/min. It was established that at 10 min of refluxing, the beta-glucosidase denatured more as the flow rate increased. At pH 2.4 and a flow rate of 10 cc/min, the activity remained constant for at least 15 min.

Effect of lidocaine on ovalbumin and egg albumin foam stability.

Foam fractionation is a simple separation process that can remove and concentrate hydrophobic molecules such as proteins, surfactants, and organic wastes from an aqueous solution. Bovine serum albumin and ovalbumin have been widely used as model proteins due to their strong foaming potential and low price. Here, we study the effect of lidocaine on albumin foam, since drugs like lidocaine are known to bind with albumin. We observed that lidocaine not only enhances the amount of foam produced but also the stability of that foam as well. The foam stability was evaluated as the decay rate constant of the foam, determined from a change in height (or volume) of the foam over a given time period.

Breathing air from protein foam.

Protein foams can be used to extinguish fires. If foams are to be used to extinguish fires where people are present, such as in high-rise buildings or ships, then a method for allowing people to breathe in a foam-filled environment is needed. It is proposed that the air, used to create the foam be used for breathing. A canister that will break incoming air-filled foam has been designed for attachment to a standard gas mask, in order to provide breathable air to a trapped person. Preliminary results for the modified mask indicate feasibility of breathing air from air-filled protein foam.

Effects of acid and alkali promoters on compressed liquid hot water pretreatment of rice straw.

In this study, effects of homogeneous acid and alkali promoters on efficiency and selectivity of LHW pretreatment of rice straw were studied. The presences of acid (0.25%v/v H2SO4, HCl, H3PO4, and oxalic acid) and alkali (0.25 w/v NaOH) efficiently promoted hydrolysis of hemicellulose, improved enzymatic digestibility of the solids, and lower the required LHW temperature. Oxalic acid was a superior promoter under the optimal LHW conditions at 160 °C, leading to the highest glucose yield from enzymatic hydrolysis (84.2%) and the lowest formation of furans. Combined with hydrolyzed glucose in the liquid, this resulted in the maximal 91.6% glucose recovery from the native rice straw. This was related to changes in surface area and crystallinity of pretreated biomass. The results showed efficiency of external promoters on increasing sugar recovery and saving energy in LHW pretreatment.

Autohydrolysis of tropical agricultural residues by compressed liquid hot water pretreatment.

Pretreatment is an essential step in biorefineries for improving digestibility of recalcitrant agricultural feedstocks prior to enzymatic hydrolysis to composite sugars, which can be further converted to fuels and chemicals. In this study, autohydrolysis by compressed liquid hot water (LHW) pretreatment of various tropical agricultural residues including sugarcane bagasse (BG), rice straw (RS), corn stover (CS), and empty palm fruit bunch (EPFB) was investigated. It was found that LHW pretreatment at 200 °C for 5-20 min resulted in high levels of hemicellulose solubilization into the liquid phase and marked improvement on enzymatic digestibility of the solid cellulose-enriched residues. The maximal yields of glucose and pentose were 409.8-482.7 mg/g and 81.1-174.0 mg/g of pretreated substrates, respectively. Comparative analysis based on severity factor showed varying susceptibility of biomass to LHW in the order of BG> RS> CS> EPFB. Structural analysis revealed surface modification of the pretreated biomass along with an increase in crystallinity index. Overall, 75.7-82.3 % yield of glucose and 27.4-42.4 % yield of pentose from the dried native biomass was recovered in the pretreated solid residues, while 18.3-29.7 % of pentoses were recovered in the liquid phase with dehydration by-product concentration under the threshold for ethanologens. The results suggest the potential of LHW as an efficient pretreatment strategy for implementation in biorefineries operated using various seasonal agricultural feedstocks.

Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica.

The sequences encoding the genes for endoglucanase II and cellobiohydrolase II from the fungus Trichoderma reesei QM9414 were successfully cloned and expressed in Yarrowia lipolytica under the control of the POX2 or TEF promoters, and using either the native or preproLip2 secretion signals. The expression level of both recombinant enzymes was compared with that obtained using Pichia pastoris, under the control of the AOX1 promoter to evaluate the utility of Y. lipolytica as a host strain for recombinant EGII and CBHII production. Extracellular endoglucanase activity was similar between TEF-preoproLip2-eglII expressed in Y. lipolytica and P. pastoris induced by 0.5 % (v/v) methanol, but when recombinant protein expression in P. pastoris was induced with 3 % (v/v) methanol, the activity was increased by about sevenfold. In contrast, the expression level of cellobiohydrolase from the TEF-preproLip2-cbhII cassette was higher in Y. lipolytica than in P. pastoris. Transformed Y. lipolytica produced up to 15 mg/l endoglucanase and 50 mg/l cellobiohydrolase, with the specific activity of both proteins being greater than their homologs produced by P. pastoris. Partial characterization of recombinant endoglucanase II and cellobiohydrolase II expressed in both yeasts revealed their optimum pH and temperature, and their pH and temperature stabilities were identical and hyperglycosylation had little effect on their enzymatic activity and properties.

A proposed mechanism for detergent-assisted foam fractionation of lysozyme and cellulase restored with beta-cyclodextrin.

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as beta-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to beta-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.