Expansion Pretreatment Research Articles

Biorefining of Lignocellulosic Feedstock by a Modified Ammonia Fiber Expansion Pretreatment and Enzymatic Hydrolysis for Production of Fermentable Sugars.

Wheat straw was pretreated and afterwards enzymatically hydrolyzed using a modified ammonia fiber expansion (AFEX) process under different reaction conditions to produce fermentable sugars. Instead of liquid ammonia, aqueous ammonia (25 % w/v) was used to test its influence on the sugar concentration and yield of the sugars. It is shown that a protein extraction after the pretreatment can distinctly improve the result obtained for the enzymatic hydrolysis. This modified AFEX process using aqueous ammonia represents a simpler and less expensive variant of the AFEX process usually described in literature. Thus, the described process can be used for the primary refining of lignocellulosic feedstocks in the sense of a roadmap for biorefinery.

Understanding the Impacts of AFEX™ Pretreatment and Densification on the Fast Pyrolysis of Corn Stover, Prairie Cord Grass, and Switchgrass.

Lignocellulosic feedstocks corn stover, prairie cord grass, and switchgrass were subjected to ammonia fiber expansion (AFEX™) pretreatment and densified using extrusion pelleting and ComPAKco densification technique. The effects of AFEX™ pretreatment and densification were studied on the fast pyrolysis product yields. Feedstocks were milled in a hammer mill using three different screen sizes (2, 4, and 8mm) and were subjected to AFEX™ pretreatment. The untreated and AFEX™-pretreated feedstocks were moisture adjusted at three levels (5, 10, and 15% wb) and were extruded using a lab-scale single screw extruder. The barrel temperature of the extruder was maintained at 75, 100, and 125°C. Durability of the extruded pellets made from AFEX™-pretreated corn stover, prairie cord grass, and switchgrass varied from 94.5 to 99.2, 94.3 to 98.7, and 90.1 to 97.5%, respectively. Results of the thermogravimetric analysis showed the decrease in the decomposition temperature of the all the feedstocks after AFEX™ pretreatment indicating the increase in thermal stability. Loose and densified feedstocks were subjected to fast pyrolysis in a lab-scale reactor, and the yields (bio-oil and bio-char) were measured. Bio-char obtained from the AFEX™-pretreated feedstocks exhibited increased bulk and particle density compared to the untreated feedstocks. The properties of the bio-oil were statistically similar for the untreated, AFEX™-pretreated, and AFEX™-pretreated densified feedstocks. Based on the bio-char and bio-oil yields, the AFEX™-pretreated feedstocks and the densified AFEX™-pretreated feedstocks (pellets and PAKs) exhibited similar behavior. Hence, it can be concluded that densifying the AFEX™-pretreated feedstocks could be a viable option in the biomass-processing depots to reduce the transportation costs and the logistical impediments without affecting the product yields.

Empty Fruit Bunch from Date Palm Industries—A Sustainable Resource for Producing Biofuels and Industrial Solvents

Abstract Date palm empty fruit bunch (EFB) is a reliable and underutilized agriculture residue generated by the date industry. EFB contains polymeric sugars that can be converted to biofuel when subjected to pretreatment, enzymatic hydrolysis, and microbial fermentation. Optimized biofuel processing conditions produced 119 kg ethanol per dry metric ton EFB. The highest sugar conversions from enzymatic hydrolysis were found with Ammonia Fiber Expansion (AFEX™, MBI International, Lansing, MI) pretreatment conditions of 2:1 ammonia-to-biomass ratio, 80% moisture content, and a temperature of 120°C, for 30 min residence time. These conditions produced 52.5 g/L and 34.8 g/L of fermentable glucose and xylose, respectively. This hydrolysate was inoculated with a glucose- and xylose-consuming recombinant yeast strain producing 35.8 g/L and 31.8 g/L ethanol with and without nutrient supplementation, respectively. A complete mass balance for EFB pretreatment, enzymatic hydrolysis, and microbial fermentation is pres...

Low pressure steam expansion pretreatment as a competitive approach to improve diosgenin yield and the production of fermentable sugar from Dioscorea zingiberensis C.H. Wright

Development of efficient pretreatment methods which can disrupt the peripheral lignocellulose and even the parenchyma cells is of great importance for production of diosgenin from turmeric rhizomes. It was found that low pressure steam expansion pretreatment (LSEP) could improve the diosgenin yield by more than 40% compared with the case without pretreatment, while simultaneously increasing the production of fermentable sugar by 27.37%. Furthermore, little inhibitory compounds were produced in LSEP process which was extremely favorable for the subsequent biotransformation of fermentable sugar to other valuable products such as ethanol. Preliminary study showed that the ethanol yield when using the fermentable sugar as carbon source was comparable to that using glucose. The liquid residue of LSEP treated turmeric tuber after diosgenin production can be utilized as a quality fermentable carbon source. Therefore, LSEP has great potential in industrial application in diosgenin clean production and comprehensive utilization of turmeric tuber.

Impact of ammonia fiber expansion (AFEX) pretreatment on energy consumption during drying, grinding, and pelletization of corn stover

ABSTRACTPretreatment and densification of biomass can increase the viability of bioenergy production by providing a feedstock that is readily hydrolyzed and able to be transported over greater distances. Ammonia fiber expansion (AFEX™) is one such method targeted for use at distributed depots to create a value-added and densified feedstock for bioenergy use. However, the pretreatment process results in a high-moisture material that must be dried, further size reduced, and pelletized, all of which are energy-intensive processes. This work quantifies the energy consumption required to dry, grind, and densify AFEX-pretreated corn stover compared to non-pretreated stover and explores the potential of reduced drying as a means to conserve energy. The purpose of this work is to understand whether material property changes resulting from AFEX pretreatment influence the material performance in downstream formatting operations. Material properties, heat balance equations, and a rotary drum dryer model were used to model a commercial-scale rotary drum dryer for AFEX-pretreated corn stover, showing the potential to reduce dryer energy consumption by up to 36% compared to non-pretreated corn stover. Laboratory-measured grinding and pelleting energies were both very sensitive to material moisture content. Overall, the total energy required for drying, grinding, and pelleting amounts to a savings of up to 23 kWh/dry Mg for the AFEX-pretreated material when dried to a low moisture content, equating to up to 0.61 $/Mg savings for gas and electricity. Grinding and pelleting of high-moisture AFEX-pretreated stover was shown to be more costlier than the savings collected through reduced drying. Although the energy and cost savings shown here are modest, the results help to highlight operational challenges and opportunities for continued improvement.

Physical and chemical characterizations of corn stalk resulting from hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment

To effectively improve enzymatic digestibility of carbohydrate in lignocellulosic biomass, hydrogen peroxide presoaking prior to ammonia fiber expansion (H-AFEX) was applied as pretreatment to corn stalk. Enzymatic hydrolysis using cocktail enzymes including cellulase, β-glucosidase and xylanase at 72h after pretreatment under optimal conditions, the glucan and xylan conversions of 88.9% and 86.3% were achieved, respectively. It was about 3.31-fold in sugar yield for H-AFEX-treated corn stalk compared with untreated material. The results of composition analysis and enzymatic hydrolysis showed that H-AFEX pretreatment was effective to remove lignin and improve glucan digestibility. The characteristics of biomass surface and cell wall, biomass crystallinity, and chemical structure changes of H-AFEX-treated corn stalk were determined by digital microscope system, X-ray diffraction and Fourier transform infrared spectroscopy (FTIR), respectively. The results demonstrated that H-AFEX pretreatment induced some morphology changes including partial damages in vascular bundle and deconstruction of cell wall, and the modified biomass structure could increase surface area accessibility and create favorable conditions for enzymatic hydrolysis obviously. Due to removal amorphous substances, the crystallinity index of H-AFEX-treated corn stalk increased comparing with that of raw material. FTIR data showed that H-AFEX process induced changes in chemical structure and cross linking such as removal/dissolution of lignin and hemicelluloses, cleavage of bonds linkage, and decrystallization of cellulose. As a result, H-AFEX pretreatment effectively reduced recalcitrance of corn stalk, and promoted subsequent enzymatic hydrolysis.

Assessment of bacterial and fungal (hemi)cellulose-degrading enzymes in saccharification of ammonia fibre expansion-pretreated Arundo donax.

This study reports enzymatic hydrolysis of the biomass of the giant reed (Arundo donax L.) after ammonia fibre expansion (AFEX) pretreatment. In particular, the capacity of the arabinofuranosidase from the fungus Pleurotus ostreatus recombinantly expressed in Pichia pastoris rPoAbf, its evolved mutant rPoAbf F435Y/Y446F and the endo-cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli to enhance the hydrolysis of AFEX-treated A. donax was investigated, using the corn stover as reference feedstock. The investigated enzymes were assayed using a mixture of purified cellulases (CBHI, CBHII, EGI and βG), endoxylanases (LX3, LX4) and accessory hemicellulases (LarbF and LβX) as reference enzyme mixture and substituting EGI with rCelStrep and LarbF with rPoAbf or rPoAbf F435Y/Y446F. The use of rPoAbf F435Y/Y446F in the substitution of LarbF led to improvements in sugar conversion, giving a glucan, xylan and arabinan conversion after 72 h of around 62, 63 and 80 %, respectively, similar or higher than those (44, 66 and 55 %) achieved by 72 h hydrolysis with commercial enzymes Novozymes Cellic®, Ctec3 and Htec3. The enzymes rPoAbf, rPoAbf F435Y/Y446F and rCelStrep were also investigated for their effect on hydrolysis of AFEX-pretreated A. donax by addition to commercial enzyme mixture Novozymes Cellic®, Ctec3 and Htec3, and it was shown that the addition of rPoAbf and its evolved mutant rPoAbf F435Y/Y446F enhanced both xylan and arabinan conversions, which achieved 80 % after 6 days of saccharification with rPoAbf F435Y/Y446F.

Effect of ammonia fiber expansion (AFEX™) pretreatment on compression behavior of corn stover, prairie cord grass and switchgrass

Understanding the fundamental mechanisms involved in densification of bulky lignocellulosic feedstocks is imperative. This study was carried out to understand the effect of ammonia fiber expansion (AFEX™) pretreatment on the compression behavior of corn stover, prairie cord grass, and switchgrass. Samples were ground using three different hammer mill screen sizes (2, 4, and 8mm) and were subjected to AFEX™ pretreatment. Untreated and AFEX™ pretreated samples were moisture adjusted to four levels (8, 12, 16, and 20% wb) and were compressed using a single pelleting unit. Physical properties comprising bulk density, particle density of the samples and unit density of pellets were determined for each combination. AFEX™ pretreated corn stover with moisture content of 20% at screen size of 2mm produced pellets with 21% higher unit density compared to untreated corn stover pellets. AFEX™ pretreated prairie cord grass and switchgrass with 20% moisture content at a screen size of 2mm produced pellets having 25% and 21% higher unit density. The decrease in hammer mill screen size and the increase in moisture content and applied pressure increased the pellet unit density. Data obtained from the compression experiments were fitted with different compaction models viz. Jones, Walker, and Kawakita and Ludde. The Kawakita and Ludde model exhibited high degree of accuracy (R2 – 0.99 and 1.00) in all the samples. The constant value ‘1/b’ in Kawakita and Ludde model represents the yield strength of the compacts, and the lower 1/b values were obtained for AFEX™ pretreated samples compared to untreated samples. This implies the impact of pretreatment, which in turn made the biomass easier to compress. Lower values of yield strength were obtained at high moisture content (16–20% wb) signifying that AFEX™ pretreated biomass at high moisture content leads to onset of deformation at relatively low pressure to produce highly compacted pellets.

Impact of Sequential Ammonia Fiber Expansion (AFEX) Pretreatment and Pelletization on the Moisture Sorption Properties of Corn Stover

Combining ammonia fiber expansion (AFEX™) pretreatment with a depot processing facility is a promising option for delivering high-value densified biomass to the emerging bioenergy industry. However, because the pretreatment process results in a high moisture material unsuitable for pelleting or storage (40% wet basis), the biomass must be immediately dried. If AFEX pretreatment results in a material that is difficult to dry, the economics of this already costly operation would be at risk. This work tests the nature of moisture sorption isotherms and thin-layer drying behavior of corn (Zea mays L.) stover at 20°C to 60°C before and after sequential AFEX pretreatment and pelletization to determine whether any negative impacts to material drying or storage may result from the AFEX process. The equilibrium moisture content to equilibrium relative humidity relationship for each of the materials was determined using dynamic vapor sorption isotherms and modeled with modified Chung-Pfost, modified Halsey, and modified Henderson temperature-dependent models as well as the Double Log Polynomial (DLP), Peleg, and Guggenheim Anderson de Boer (GAB) temperature-independent models. Drying kinetics were quantified under thin-layer laboratory testing and modeled using the Modified Page's equation. Water activity isotherms for non-pelleted biomass were best modeled with the Peleg temperature-independent equation while isotherms for the pelleted biomass were best modeled with the Double Log Polynomial equation. Thin-layer drying results were accurately modeled with the Modified Page's equation. The results of this work indicate that AFEX pretreatment results in drying properties more favorable than or equal to that of raw corn stover, and pellets of superior physical stability in storage.

Comparison of Hydrogen Peroxide and Ammonia Pretreatment of Corn Stover: Solid Recovery, Composition Changes, and Enzymatic Hydrolysis

Three pretreatments, hydrogen peroxide pretreatment (HP), ammonia fiber expansion pretreatment (AFEX), and hydrogen peroxide presoaking prior to AFEX (H-AFEX), were employed to improve enzymatic di...

Enzymatic hydrolysis of pelletized AFEX™‐treated corn stover at high solid loadings

Ammonia fiber expansion (AFEX™) pretreatment can be performed at small depots, and the pretreated biomass can then be pelletized and shipped to a centralized refinery. To determine the feasibility of this approach, pelletized AFEX-treated corn stover was hydrolyzed at high (18-36%) solid loadings. Water absorption and retention by the pellets was low compared to unpelletized stover, which allowed enzymatic hydrolysis slurries to remain well mixed without the need for fed-batch addition. Glucose yields of 68% and xylose yields of 65% were obtained with 20 mg enzyme/g glucan and 18% solid loading after 72 h, compared to 61% and 59% for unpelletized corn stover. Pelletization also slightly increased the initial rate of hydrolysis compared to unpelletized biomass. The ease of mixing and high yields obtained suggests that pelletization after AFEX pretreatment could have additional advantages beyond improved logistical handling of biomass.

Application of new expansion pretreatment method on agricultural waste. Part I: Influence of pretreatment on the properties of lignin

In the present work, a new expansion pretreatment was used to fractionate agricultural wastes (rice straw and rice husk) into lignin and carbohydrates. The expansion pretreatment takes the advantages of extrusion and milder steam explosion. The lignin fractions obtained from expanded rice straw and rice husk by mild alkaline extraction were characterized by gel permeation chromatography (GPC), sugar analysis, 2D NMR and derivatization followed by reductive cleavage (DFRC). Results showed that the expansion pretreatment resulted in higher extraction yields of lignin from pretreated rice straw than that from untreated control. The results from molecular weight measurement and DFRC analysis suggested that the condensation reaction could take place between lignin units during the expansion processing. 2D NMR spectra showed that guaiacyl (G) and syringyl (S) units were predominant in the lignin fractions, and a small amount of p-hydroxyphenyl (H) unit was also detected. It was also found that the flavones-type structure tricin was apparently present in lignin fractions from rice straw but not in rice husk. Considering the high extraction yields and purity of lignins from pretreated biomass, the expansion pretreatment could be an attractive method for fractionation of agricultural wastes.

Novel method utilizing microbial treatment for cleaner production of diosgenin from Dioscorea zingiberensis C.H. Wright (DZW)

A novel method utilizing microbial treatment for cleaner production of diosgenin from Dioscorea zingiberensis C.H. Wright (DZW) was presented. A new Bacillus pumilus HR19, which has the great ability to secrete pectinase, was screened and applied in the microbial treatment. Low-pressure steam expansion pretreatment (LSEP) was employed in advance to assist microbial treatment efficiently in releasing saponins, which are the precursors of diosgenin. Compared with the traditional process of acid hydrolysis, this novel process reduced the consumptions of water, acid and organic solvent by more than 92.5%, 97.0%, 97.0%, respectively, while simultaneously increasing the diosgenin yield by 6.21%. In addition, the microbial treatment was more efficient than enzymatic treatment, which arised from that microorganisms could be induced to secrete related enzymes by the compositions of DZW and relieve product inhibition by utilizing enzyme hydrolysates.

Recycled aqueous ammonia expansion (RAAE) pretreatment to improve enzymatic digestibility of corn stalks

A novel lignocellulose pretreatment method using aqueous ammonia for biofuel production was proposed in this study, which named recycled aqueous ammonia expansion (RAAE). Effects of temperature, pretreatment time, water to dry corn stalks loading and flow rate of aqueous ammonia on substrate enzymatic digestibility and sugar yield were investigated. Pretreatment temperature and time are important factors that affect RAAE process. Recirculation process could improve biomass digestibility and sugar yield compared with batch experiment. After RAAE pretreatment, about 70% of the lignin was removed, while more than 90% of the cellulose was preserved in the solids, the substrate crystallinity also increased because of the removal of amorphous portion. The maximum glucan enzymatic digestibility of pretreated biomass was 85.70%, which was obtained at 85°C, 11 min, 80% water to dry corn stalks loading and 1.5L/min aqueous ammonia flow rate.

Application of corncob-based activated carbon as electrode material for electric double-layer capacitors

To investigate the influence of expansion pretreatment for materials on carbon structure, activated carbons (ACs) were prepared from corncob with/without expansion pretreatment by KOH activation, the structure properties of which were determined based on N2 adsorption isotherm at 77 K. The results show that the expansion pretreatment for corncobs is beneficial to the preparation of ACs with high surface area. The specific surface area of the AC derived from corncob with expansion pretreatment (AC-1) is 32.5% larger than that without expansion pretreatment (AC-2). Furthermore, to probe the potential application of corncob-based ACs in electric double-layer capacitor (EDLC), the prepared ACs were used as electrode materials to assemble EDLC, and its electrochemical performance was investigated. The results indicate that the specific capacitance of AC-1 is 276 F/g at 50 mA/g, which increases by 27% compared with that of AC-2 (217 F/g). As electrode materials, AC-1 presents a better electrochemical performance than AC-2, including a higher voltage maintenance ratio and a lower leakage current.

Influence of variable species composition on the saccharification of AFEX™ pretreated biomass from unmanaged fields in comparison to corn stover

Abstract From the perspective of the biorefinery, the inherent heterogeneity of mixed-species feedstocks increases the apparent risk associated with their use due to the difficulty in predicting processing characteristics, potential yields and digestibility. These materials often contain species from different botanical classifications, which could affect pretreatment efficiency and Saccharification yields. For this study, we evaluated the impact of ammonia fiber expansion (AFEX™ 1 ) pretreatment conditions on hydrolysis sugar yields for five early successional old-field treatment replicates, each comprised of a different mixture of annual forb and grass species. Yields from these samples were also compared to a late successional old field sample and to corn stover that had been pretreated at the same conditions. Relatively high sugar yields were obtained for four of the five early successional feedstocks when pretreated at the same conditions: 2.0 g NH3:g DM; 0.5 g H2O:g DM; 100 °C; 30 min. It was found that glucan digestibility was strongly inversely correlated to the total lignin content; however this was not true of the xylan digestibility. The mixed-species feedstocks that had a higher grass content tended to also have a higher structural sugar content and were more digestible than forb-dominated feedstocks, thereby resulting in higher saccharification yields. Also, the grass-dominated mixed-species feedstocks were as digestible as corn stover (∼80% total sugar yields). However, due to its higher structural sugar content, corn stover had higher total glucose and xylose yields (580 g/kg biomass) compared to the mixed-species feedstocks, which ranged from 290 to 470 g/kg biomass.

Guayule as a feedstock for lignocellulosic biorefineries using ammonia fiber expansion (AFEX) pretreatment

Natural rubber latex extraction from guayule leaves behind greater than 90% (by weight) of agricultural residue as a feedstock suitable for conversion to biofuels via a thermochemical or biochemical route. Untreated guayule shrub and bagasse (after latex extraction) has shown to be very recalcitrant to enzymatic hydrolysis, necessitating application of a chemical pretreatment to enhance cellulase accessibility. The objective of this work was to carry out detailed compositional analysis, ammonia fiber expansion (AFEX11AFEX: ammonia fiber expansion.) pretreatment, enzymatic hydrolysis and ethanol fermentation for various guayule-derived biomass fractions. Plant feedstocks tested were derived from two sources; (a) a mature 2007 AZ-2 whole guayule shrub plant obtained from USDA/ARS22USDA/ARS: United States Department of Agriculture/Agricultural Research Service. research fields, and (b) the guayule latex-extracted commercial grade bagasse (62505) from Yulex Corporation. Compositional analysis and enzymatic hydrolysis were carried out using standard NREL33NREL: National Renewable Energy Laboratory. protocols (www.nrel.gov/biomass/analytical_procedures.html). AFEX pretreatment was carried out using concentrated ammonium hydroxide at elevated temperatures for desired residence times in a pressurized reactor. Yeast fermentations on biomass hydrolyzates were carried out micro-aerobically using Saccharomyces cerevisiae (424A strain) in shake flasks.AFEX pretreatment was found to substantially improve overall enzymatic digestibility by 4–20 fold for both untreated guayule shrub and latex-extracted bagasse. Maximum glucan and xylan conversion achieved for the latex-extracted bagasse was 40% and 50%, respectively. The yeast was readily able to ferment both glucose and xylose to ethanol from the guayule bagasse hydrolyzate with or without external nutrient supplementation (i.e., yeast extract and tryptone). Our results highlight the possible utilization of guayule as a feedstock for lignocellulosic refineries co-producing natural rubber latex and biofuels. However, further process improvements (e.g., lignin/resin extraction and cellulose decrystallization using a modified AFEX process) are necessary to increase the effectiveness of ammonia-based pretreatments for further enhancing enzymatic digestibility of guayule-derived hardwood biomass.

Low Temperature and Long Residence Time AFEX Pretreatment of Corn Stover

Low temperature and long residence time pretreatments have been proposed as an alternative to conventional pretreatments within a centralized biorefinery, allowing for a decentralized pretreatment without high energy costs. Ammonia fiber expansion (AFEX™) pretreatment may be uniquely suitable for decentralized pretreatment, and this study considers the possibility of decreasing the temperature in AFEX pretreatment of corn stover. AFEX pretreatment at 40°C and 8 h produced comparable sugar and ethanol yields as conventional AFEX pretreatment at high temperatures and short residence time during subsequent hydrolysis and fermentation. Increasing the ammonia loading at these temperatures tends to increase digestibility, although the moisture content of the reaction has little effect. This study suggests a greater flexibility in AFEX pretreatment conditions than previously thought, allowing for an alternative approach for decentralized facilities if the economic conditions are appropriate.

AFEX Pretreatment and Enzymatic Conversion of Black Locust (Robinia pseudoacacia L.) to Soluble Sugars

Black locust (Robinia pseudoacacia L.), like willow and poplar, is a hardwood species which can be grown in coppice for bioenergy production, and because of its nitrogen-fixing ability, it can be cultivated with higher yields on less productive land. For these experiments, we examined the feasibility of using ammonia fiber expansion (AFEX) pretreatment to increase the saccharification yields from black locust grown for bioethanol production, as well as examine the impact of posttreatments (hot-water washing and additional size reduction) on sugar yields. The optimal AFEX conditions for black locust were 180°C, 1.0 g NH3/g dry biomass, 2.5 g H2O/g dry biomass, for 30 min residence time, and of the parameters tested, temperature had the greatest impact on yields. Yields from the sample without posttreatment and hydrolyzed at the standard enzyme loading were very low: <30% glucose and ∼50% hemicellulose. Both hot-water washing and size reduction improved yields; however, size reduction had a more significant effect indicating that increasing enzyme accessibility is more important for digestibility as opposed to the removal of soluble inhibitors. The effect of size reduction was comparable to that obtained by quadrupling the enzyme loading, increasing glucose yields by ∼20–30% and hemicellulose yields by ∼20%. Untreated black locust is known to contain compounds which are inhibitory to both enzymes and microorganisms and AFEX pretreatment neutralizes this inhibitory effect to some extent.

Effect of Extruding Parameters on Soybean Oil Yield of Enzyme-Assisted Aqueous Extraction Processing

Experiments were carried out to investigate effects of extruding parameters on soybean oil yield extracted with enzyme-assisted aqueous extraction. It was found that extrusion and expansion parameters affected significantly on total soy oil yield. Optimal extrusion and expansion parameters for highest total soy oil yield were obtained using response surface methodology. Effect of extrusion and expansion pretreatment on soy cells was studied using light microscope and transmissive microscope. State of free oil and protein during hydrolysis was observed using light microscope. Ultrasonic assistant ethanol de-emulsification was found the effective method to destabilize the emulsion.