A tissue-mimicking phantom that accurately represents human-tissue properties is important for safety testing and for validating new imaging techniques. To achieve a variety of desired human-tissue properties, we have fabricated and tested several variations of gelatin phantoms. These phantoms are simple to manufacture and have properties in the same order of magnitude as those of soft tissues. This is important for quality-assurance verification as well as validation of magnetic resonance-guided focused ultrasound (MRgFUS) treatment techniques.1 and T2*), mechanical (density and Young’s modulus), and thermal properties , were obtained through independent measurements for all three bloom types to characterize the gelatin phantoms. Thermal repeatability of the phantoms was also assessed using MRgFUS and MR thermometry.The phantoms presented in this work were constructed from gelatin powders with three different bloom values , each one allowing for a different mechanical stiffness of the phantom. Evaporated milk was used to replace half of the water in the recipe for the gelatin phantoms in order to achieve attenuation and speed of sound values in soft tissue ranges. These acoustic properties, along with MR  sonications, interleaved with high-power (up to 22.0 W) sonications, each of the three different bloom phantoms demonstrated repeatable temperature increases  for heating durations of 18.1 s.These evaporated milk-modified gelatin phantoms should serve as reliable, general soft tissue-mimicking MRgFUS phantoms. Magnetic resonance-guided focused ultrasound (MRgFUS) is a promising, emerging technology that has been utilized as a thermal therapy with applications that include the treatment of uterine fibroids , 2, prosThere are several commercial phantom options available for MRgFUS therapies, such as those by ATS  and CIRS . Commercial phantoms that are made consistently with controlled properties allow direct comparison of results from multiple groups that use the same product. Furthermore, commercial phantoms often have published values for several properties. While commercial phantoms are good options for many applications, their properties are not easily changed for specific applications. Custom making a phantom in-house provides an option for fabricating phantoms with shapes and properties tailored to the application and tissue of interest. The drawback in custom fabrication is the potential variability between batches, which may make experimental repeatability difficult. The purpose of this work was to fabricate a simple gelatin phantom suitable for both thermal ablation and ARFI testing and to characterize it by measuring its acoustic, MR, mechanical, and thermal properties, while also comparing it with soft tissue values of interest, such as in muscle, fat, breast, and brain tissues.All phantoms were made using porcine gelatin powder of either 250-bloom , 175-bloom , or 125-bloom (Vyse). The bloom value is defined by the Gelatin Manufacturers Institute of America by measuring the weight (in grams) necessary to displace a 0.5-in diameter piston 4 mm into the solid gelatin, made with 6.67 % gelatin, 17 h after production and at a temperature of 10 °C . Due to 1 In this study, it was found that both the speed of sound and attenuation of the gelatin increased as the percentage of evaporated milk to water was increased during fabrication, as shown in Table The method for fabricating phantoms described herein was modified from the standard ballistics gelatin recipe  in that To create each liter of the 50 % water/50 % milk gelatin phantom, 111 grams of gelatin powder with one of the three different bloom values  is mixed with 225 mL of de-ionized degassed water in a sanitized container that needs to be capable of containing the final volume of the phantom. Though the standard definition of bloom is based on 6.67 % gelatin, we used the 11.1 % recommended by the gelatin manufacturer. Ten drops of Vyse defoamer solution  are added after the powder and water have been mixed. In a separate sanitized container, 275 mL of degassed de-ionized water is mixed with 500 mL of evaporated milk. The evaporated milk/water mixture is heated to 80 °C, then poured into the gelatin/water mixture where it is thoroughly stirred until all the gelatin is fully dissolved, resembling a uniform liquid. This mixture is allowed to cool to 40 °C before pouring it through a strainer into the final (sanitized) phantom mold.In our studies, no preservatives were added to the phantoms, but if desired, this could be added to the uniform gelatin/milk liquid mixture before it cooled to 40 °C. Once the gelatin was poured into its mold  at the start of each waveform  and at the end , to isolate the sample from noise. A line was fitted to each attenuation value found at each frequency using linear least squares method in which the slope of the line provided the final attenuation value assigned to the sample in dB/cm/MHz.The attenuation  and multi-echo gradient recalled echo (ME GRE) sequences, respectively. For all measurements, care was taken to optimize the shimming. The imaging parameters for the STIR sequence were as follows: repetition time (TR) 6000 ms, echo time (TE) 17 ms, inversion recovery times (TI) between 50 to 2500 ms , field of view (FoV) 200 × 128 mm, resolution (Res) 1.6 × 1.6 × 3.0 mm, flip angle (FA) 90/180°, and bandwidth (BW) 240 Hz/pixel. For the ME GRE sequence, the parameters were the following: TR 90 ms, TE between 2.83 to 80 ms , FoV 192 × 144 mm, Res 0.8 × 0.8 × 5.0 mm, FA 20°, and BW 810 Hz/pixel.Phantom MR properties were measured on a Siemens 3T MRI scanner . One phantom was tested for each bloom value using the mold shown in Fig. A single-loop single-channel RF receive-only coil that was custom-built in-house for the phantom molds was used to detect the signal. The coil’s diameter was slightly larger than the 11.5-cm outer diameter of the cylindrical phantom holders . For mechanical property characterization, three batches were made for each gelatin-bloom value. Each batch of a given bloom value yielded multiple disk specimens uniformly created with a machined metal punch. Each disk had a diameter of 20 mm and a height of 10 mm; six sample disks were measured per batch. A frictionless boundary was achieved by applying canola oil to the surface of both compression plates. The top compression disk had a diameter of 22 mm, while the bottom plate’s diameter was 76 mm. A total strain of 15 % was performed (1.5-mm compression) on each disk in 2 s . Instron’s Bluehill 3 software was used to acquire the measured force and displacement, which in turn were used to calculate Young’s modulus. The Young’s modulus was averaged for the six disks in each batch, then averaged across the three batches. This was done for each bloom value .Density was calculated by measuring the mass of each disk, then by measuring the volume of water displaced by each sample disk in a graduated cylinder. Six sample disks were measured from one batch for each of the three gelatin-bloom values, and then averaged to yield the density of each bloom value.ψ) and thermal conductivity (k) were determined with a KD2 Pro invasive thermal probe . For each gelatin-bloom value, three measurements were obtained at different locations within a single phantom (selected from those used for acoustic measurements) and then averaged. The two-prong probe was inserted 3 cm into the phantom sample, where it remained undisturbed for approximately 3 min.Thermal diffusivity (cp) was calculated using Eq. ρ) from the mechanical tests was used for each bloom value, but the three individual thermal diffusivity and thermal conductivity values measured for each sample with the KD2 Pro were used to calculate three separate specific heat capacities per bloom value, which were then averaged.The specific heat capacity -shift MR-thermometry technique . A 1-MHzAll heating was done with the geometric focus positioned 3 cm into the phantoms. A fiber optic temperature probe  was inserted 4 cm into the other side of the phantom to measure the bulk temperature of the gelatin, approximately 8 cm away from the beam focus; all phantoms started at the MR suite’s ambient temperature (~24 °C).The 3D MRI temperatures in the phantoms were obtained using a segmented GRE echo planar imaging (EPI) pulse sequence with TR 25 ms, TE 13 ms, FoV 192 × 96 × 32 mm, Res 1.2 × 1.2 × 2.0 mm (zero-filled interpolated to 0.5-mm isotropic voxel spacing), number of slices 16, FA 20°, BW 744 Hz/pixel, EPI factor 9, echo spacing 1.59 ms, acquisition time 3.625 s, with no fat saturation pulse applied. The start of each 18.125 s ultrasound-heating exposure was synchronized with the beginning of the sixth MR measurement using a fiber optic trigger pulse emitted by the pulse sequence. Between each heating exposure was a 10-min cooling period. Based on our previous experience with MR-temperature measurements in similar phantoms, the 10-min minimum-cooling interval was found sufficient to allow the heated region of the phantoms to return to a baseline-temperature value.n” refers to the number of times a test was performed within the same batch of gelatin, while “N” refers to the number of times a test was performed on separate batches of gelatin. The intra-batch standard deviation for T1, T2*, density, thermal diffusivity, and specific heat capacity is reported in Table Table Thermal repeatability was tested for phantoms of each bloom value, and the temperature responses at the location of peak temperature for the 125-bloom phantom are displayed in Fig. For the 125-bloom phantom, the five 6.6-W low-power exposures, interspersed with the higher power sonications, produced peak temperature increases of 10.4 ± 0.3 °C. The interspersed medium-power exposure of 13.2 W yielded a temperature rise of 19.8 °C, and the high-power exposure of 20.7 W had a temperature rise of 26.7 °C. To analyze the temperature-measurement uncertainty, the standard deviation through time  for each of 20 × 20 × 20 voxels  in an unheated region of the phantom was calculated. The mean and standard deviation of these 8000 standard deviation values was 0.20 ± 0.03 °C, demonstrating the good precision in the temperature measurements.For the 175-bloom phantom, the five 6.6-W low-power exposures produced temperature increases of 10.2 ± 0.3 °C. The interspersed medium-power sonication of 13.2 W yielded a temperature rise of 19.6 °C, and the 20.7-W high-power sonications had a temperature rise of 28.2 ± 0.2 °C. The temperature standard deviation through time of an unheated 20 × 20 × 20 voxel region of the 175-bloom gelatin phantom was 0.23 ± 0.04 °C.For clarity, not all sixteen heating runs performed in the 250-bloom phantom are shown in Fig. In this study, the acoustic, MR, mechanical, and thermal properties for gelatin phantoms with three bloom values have been characterized using independent methods. These easy-to-make gelatin phantoms provide properties of the same order of magnitude as soft tissues, without precisely mimicking a particular tissue. However, it is also important to acknowledge that each tissue type is not a homogeneous medium, and these phantoms cannot mimic the typical heterogeneity of a specific tissue type that has a range of valid values present. Despite this limitation, these gelatins potentially allow a user to customize the phantom further to match the particular homogeneous properties of a specific soft tissue.The nominal requirements for soft tissue-like acoustic properties developed over the years by the ultrasound phantom research community , 20–24 iThe top two rows of Table 1 and T2* literature values were measured in white matter for the brain and in glandular tissue for the breast. The fat T1 literature value was measured in breast fat. All three of the gelatin values have similar MR properties that fall between the literature values for the listed soft tissues. The 250-bloom gelatin phantom has both a T1 value and a T2* value very similar to brain white matter. If desired, the T1 values of the phantoms can be decreased by adding various amounts of copper sulfate to the gelatins during construction [Also in Table truction , though Table The last two rows of Table These gelatin phantoms demonstrated thermal repeatability when heated with MRgFUS using low-power heatings interspersed with medium- to high-power heatings, as summarized in Table 1 and T2*) for these gelatin phantoms will need further study.The thermal repeatability tests showed that the magnitude and position of the temperature rise was predictable in 31 of 32 cases. Only for the highest power exposure (22.0 W) with the 125-bloom sample was there a deviation from the time and temperature patterns seen in previous exposures. The location of the peak temperature also shifted one voxel deeper into the phantom than measured in all other exposures. This may be due to spot melting of the gelatin at the focus; however, we did not do independent verification of this. Future studies will investigate the effects that cross-linkers have on the gelatin phantoms by re-testing their thermal repeatability. By adding cross-linkers to the gelatin, the temperature at which the gelatin phantoms melt can be increased , 37. ThoOur group has employed the homogeneous soft tissue-mimicking phantoms described in this work for both thermal ablation evaluations and ARFI. All three bloom values with their varying Young’s moduli are useful for ARFI experiments and have been used in validating ARFI simulations with good correlation to experimental results . Also, pThis work provides a general soft tissue, repeatable gelatin-phantom recipe that is easy to manufacture. Several of these simple gelatin phantoms have been characterized for acoustic, MR, mechanical, and thermal properties, and have been shown to demonstrate thermal repeatability. Future studies will entail adding cross-linkers to these phantoms to increase their melting temperatures and investigate the effect on other phantom properties. Also, future work can investigate the interrelationship between other additions and subsequent changes to the phantom properties. By exploring such modifications to the gelatin recipe, one may be able to adjust the gelatin to better match a specific property for a soft tissue of interest.1Vegetable glycerin was also tested to replace water volume in the gelatin recipe in order to increase the phantom’s viscosity as a potential option for increasing the attenuation. However, the speed of sound increased more drastically than with evaporated milk with only a small increase in attenuation. Also, the glycerin was more difficult to keep degassed as air bubbles were visible in the final solidified gelatin phantoms.


We demonstrate theoretically and experimentally an interesting opaque state, which is based on an analog of electromagnetically induced transparency (EIT) in mechanism, in a metal hole array of the dimer lattice. By introducing a small difference to the dimer holes of each unit cell, the surface delocalized modes launching out from the dimer holes can have destructive interferences. Consequently, a narrow opaque window in the transparent background can be observed in the transmission spectrum. This surface-mode-induced opacity (SMIO) state is very sensitive to the difference of the dimer holes, which will promise various applications. Electromagnetically induced transparency (EIT) is an interesting phenomenon for both scientific and technological aspects due to its intriguing properties such as reducing the group velocity of the light256791011121314151617181920212223242526282911182829In this work we consider metal hole arrays313220211 = ε2 = 1), a small difference in the dielectric constants of the dimer holes  leads to an EIT-like spectrum  of dimer lattice is schematically shown in the inset of 32341 = 1.21 holes (2 = 1 holes at the higher transmission peak (1 = ε2 = 1) and show it in To confirm the above understanding, we calculate the tangential electric field  on the metal surface at the two transmission peaks and the opaque state. As one expected, at the lower transmission peak great field enhancement is observed around the ε21 holes , while tion peak . At the ion peak . For a cg of the dimer holes .


The measurement of arterial stiffness has long been a reliable method of determining the severity and risk involved with many cardiovascular diseases . Magneti2, matrix = 128×64, slice thickness = 6 mm, #slices = 3, #segments = 7-8, 8 cardiac phases, and a motion encoding gradient (MEG) ranging from 90-100 Hz applied separately in the x, y, and z directions. Sagittal images were masked to delineate the abdominal aorta . Volunteers were positioned head first in the supine position in the scanner. 70 Hz mechanical waves were introduced into the aorta using a pneumatic driver system . A 2D seFigure This preliminary study demonstrated the feasibility of determining aortic stiffness values during the cardiac cycle and also showed that aortic stiffness values varied during the cardiac cycle. The stiffness values are lower during the diastolic phases when compared to systolic phases. However, additional data is warranted to establish the stiffness values of the aorta during the cardiac cycle.The Ohio State University Department of Radiology.


Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.Although wheat ( Wheat is one of the most important cereal crops cultivated worldwide. Wheat caryopsis contains an abundance of nutrients, including starch, proteins, minerals, vitamins, and phytochemicals . Starch Starch is composed of two major glucose polymers, namely, amylose and amylopectin, which differ in their degree of polymerization of glucan chains . The keySorghum bicolor (L.) pericarp consist of multiple starch granules; they present high accumulations in the mesocarp at early stages of caryopsis development but eventually disappear at maturity [In cereal crop endosperm cells, starch granules are synthesized in the amyloplast and increase in number and volume until caryopsis maturity , 12. Thematurity . In ricematurity . In wheamaturity . Similarmaturity , who poimaturity  studied AGPase, GBSS II, and AMY), and degradation mechanisms of PSGs remains limited. In our previous study, the development and physicochemical properties of PSGs and endosperm starch granules were compared [Although the accumulation characteristics of PSGs are well-studied, other characteristics, such as starch granule development in different pericarp regions as well as the structural characteristics and physicochemical properties of PSGs in developing wheat caryopsis, remain poorly understood. Moreover, our knowledge of the amylose content, relative expression of the related synthetic and degradation enzyme genes (compared . Based oTriticum aestivum L. cv. Yannong 19) seeds used in the present study were purchased from the National Wheat Improvement Center  and grown in the experimental field of Yangzhou University , Key Laboratory of Crop Genetics and Physiology of Jiangsu Province from September 2013 to May 2014. The field soil was sandy loam ) that contained 24.5 g kg-1 organic materials as well as 106, 33.8, and 66.4 mg kg-1 of available N, P, and K, respectively. The climate condition represented a transition from humid subtropical monsoon climate to temperate monsoon climate. The average annual temperature was 15.2°C and the annual rainfall was 961–1048 mm. The plants received normal fertilizer and water supply during growing stages. To determine the anthesis date accurately, individual florets were marked at the base of the spikelet in the middle of spikes and whole plants were tagged.The high-yielding hard winter wheat   with sliThe separated pericarp was dried in an oven at 110°C for 3 h and completely ground in a mortar. The powder obtained was used to determine total starch and soluble sugar contents via total starch  and soluble sugar  kits. Amylose contents in the extracted starch were determined using an amylose determination kit .4 for 3 h. Fixed samples were subsequently rinsed, dehydrated, infiltrated, and embedded according to the method described by Zheng and Wang (2011) [Caryopses were collected 2, 6, 10, and 20 DAA and cut breadthwise into 2 mm-thick slices from the center. Samples were immediately immersed in 0.1 M glutaraldehyde-phosphate buffer fixative (pH 7.2) at 4°C for 3 h and then fixed with 0.5% OsOFor light microscopy, samples were cut into 1 μm slices using an ultramicrotome , stained with 0.5% methyl violet for 10 min, and photographed under a light microscope .For transmission electron microscopy, samples were cut into 60 nm slices using an ultramicrotome , respectively stained with uranyl acetate and lead citrate for 30 and 15 min, and photographed under a transmission electron microscope .For fluorescence microscopy, caryopses were collected and cut breadthwise into 1 mm-thick slices from the center. Samples were placed on a microslide and observed and photographed under a fluorescence microscope  equipped with a digital camera .For scanning electron microscopy, 1 mg of starch was mixed with 500 μL of absolute ethyl alcohol. The mixture (20 μL) was placed in a stage groove and dried at 40°C for 1 h. Samples were then gold-sputtered under vacuum and photographed using a scanning electron microscope . The accelerating voltage and current of the instrument were 15 kV and 10 μA, respectively. The size distribution of starch granules in the micrographs was analyzed using Image-Pro Plus software . A total of 200 starch granules were combined into one sample, and the means of three replicate samples  were used for calculations.XRD was used to investigate the crystalline structure of starch granules including the crystalline type and relative degree of crystallinity. Prior to determination, starch samples were stored in a closed container containing 100 mL of saturated NaCl solution maintained under a constant-humidity atmosphere (relative humidity = 75%) for 7 d. Wet starch samples were placed on a vitric sample platform and scanned at diffraction angles (coupled 2θ) from 4° to 40° with a step size of 0.2 s. The ratio of the crystallinity area to the total diffraction area was calculated as the relative degree of crystallinity (%) from the XRD patterns obtained using Image-Pro Plus software  .−1 to 800 cm−1 and then deconvoluted with a resolution enhancement factor of 1.9 and a half-width of 19 cm−1 [−1 were measured by recording the height of the bands from baseline to peak point.An FTIR spectrometer equipped with an attenuated total reflectance (ATR) system was used to analyze ordered structures in the external region of the starch granules . Starch  19 cm−1 . Infrare2, 0.02% NaN3, and 50 U PPA) [g for 10 min at 4°C. To determine the degree of hydrolysis, soluble sugar contents in the supernatant were measured using a soluble sugar kit .Starch was hydrolyzed using porcine pancreatic α-amylase . Starch samples (10 mg) were suspended in 2 mL of enzyme solution  . HydrolyRNAprep pure plant kit . Each sample (5 mg) was reverse transcribed into cDNA using the PrimeScript RT reagent kit  following the manufacturer’s instructions. PCR amplifications were performed using primer pairs  and used to transform the E. coli strain DH5α. After PCR identification of the extracted plasmid, DNA sequencing was performed using GenScript  (S1 Table).The pericarp was separated from the caryopsis by hand at 2, 6, 10, and 20 DAA. The separated pericarp was immediately frozen in liquid nitrogen for 5 min and then stored at –70°C until RNA isolation. Total RNA was extracted from the pericarp using an er pairs  to obtaiADP-ribosylation factor gene), which is considered one of the most stably expressed genes [qPCR analysis was carried out on a qPCR system  with SYBR Premix Ex Taq (TaKaRa). Wheat Ta2291 . Means were compared using Fisher’s protected least significant difference test at the 0.05 probability significance level.At 2 DAA, a large number of starch granules accumulated in different regions of the pericarp, including the epicarp, mesocarp, and MCVB . New celAt 6 DAA, apoptosis cavities  than earlier stages (2 and 6 DAA). By contrast, short-range ordered structures in the external region of the granules did not exhibit changes with pericarp development.Amylose and short chains of amylopectin formed a double helix structure (long-range ordered stucture). Meanwhile, these double-helical molecules held the crystalline stucture (short-range ordered stucture) within starch granules via intermolecular interaction forces. PSGs at different developmental stages showed similar ATR-FTIR spectra . No signobserved . The IR AGPase, GBSS II, and AMY in wheat pericarp at different developmental stages  were examined by qPCR, and patterns of relative gene expression are shown in AGPase gene, expression increased from 2 DAA to 6 DAA and gradually decreased thereafter  . SimilarAMY gene shows significantly higher relative expression at 6 DAA than at 2 DAA. Laethauwer et al. (2013) [AMY gene expression in winter types of triticale. Thus, the AMY gene product α-amylase likely catalyzes the hydrolysis of starch granules from 6 DAA to 20 DAA.PSGs began to appear at 6 DAA and then gradually disappeared until 20 DAA. This phenomenon may be explained in two ways: (1) from 6 DAA to 20 DAA, endosperm cells undergo a period of material enrichment, including protein body and starch granule synthesis  Fig 3)..Fig 3). . (2013)  reportedAGPase gene, which activates the first step of starch biosynthesis and shows increases from 2 DAA to 6 DAA and decreases from 6 DAA to 20 DAA. From 6 DAA to 10 DAA, soluble sugar contents increased rapidly and starch granule hydrolysis could be observed. From 10 DAA to 20 DAA, both total starch and soluble sugar contents decreased, which indicates that these carbohydrates are not only responsible for pericarp survival but also provide additional nutrients for endosperm growth. Similar ideas have been proposed previously by Yu et al. (2015) [Dynamic changes in total starch and soluble sugar contents reveal that decreases in soluble sugar contents from 2 DAA to 6 DAA may support starch synthesis, which results in increases in total starch content. These results are consistent with the relative expression of the . (2015)  and Zhou. (2015) .GBSS II gene showed higher relative expression from 2 DAA to 6 DAA than at later stages, indicating that the activity of amylose synthesis was greater at early stages than that at later stages. When starch was hydrolyzed by amylase, the amorphous region containing amylose was degraded first, followed by the crystalline region containing amylopectin molecules [GBSS is the only starch synthase that is found exclusively within starch granules and controls amylose synthesis . GBSS IIolecules . This maPSGs were found to have not only large granule sizes but also rough granule surfaces, which could be caused by α-amylase attack. When hydrolyzed by α-amylase, the degree of enzymolysis of PSGs followed the order: 2 DAA>6 DAA>10 DAA>20 DAA. These results could be due to the ratio of surface area to volume of granules, since previous studies demonstrated that starch granules with smaller sizes show higher degrees of enzymolysis . In this−1 are associated with crystalline lamellar and amorphous structures, respectively, while the band at 995 cm−1 is attributed to the bending vibrations of C–OH bonds [−1/1022 cm−1 and 1022 cm−1/995 cm−1 are considered quantitative indices for evaluating the short-range ordered structures of starch granules [XRD and ATR-FTIR are advanced technologies that have been successfully used to study long-range ordered structures of whole granules and short-range ordered structures of external regions of starch granules, respectively , 38. TheOH bonds . The IR granules . In thisS1 TableAdditional supporting information may be found in the online version of this article at the publisher’s web-site.(DOCX)Click here for additional data file.


Oil palm trunk (OPT) is a valuable bioresource for the biorefinery industry producing biofuels and biochemicals. It has the distinct feature of containing a large amount of starch, which, unlike cellulose, can be easily solubilized by water when heated and hydrolyzed to glucose by amylolytic enzymes without pretreatment for breaking down the biomass recalcitrance. Therefore, it is suggested as beneficial to extract most of the starch from OPT through autoclaving and subsequent amylolytic hydrolysis prior to pretreatment. However, this treatment requires high capital and operational costs, and there could be a high probability of microbial contamination during starch processing. In terms of biochemical conversion of OPT, this study aimed to develop a simple and efficient ethanol conversion process without any chemical use such as acids and bases or detoxification.For comparison with the proposed efficient ethanol conversion process, OPT was subjected to hydrothermal treatment at 180 °C for 30 min. After enzymatic hydrolysis of PWS, 43.5 g of glucose per 100 g dry biomass was obtained, which corresponds to 81.3 % of the theoretical glucose yield. Through subsequent alcohol fermentation, 81.4 % ethanol yield of the theoretical ethanol yield was achieved. To conduct the proposed new process, starch in OPT was converted to ethanol through enzymatic hydrolysis and subsequent fermentation prior to hydrothermal treatment, and the resulting slurry was subjected to identical processes that were applied to control. Consequently, a high-glucose yield of 96.3 % was achieved, and the resulting ethanol yield was 93.5 %.The proposed new process was a simple method for minimizing the loss of starch during biochemical conversion and maximizing ethanol production as well as fermentable sugars from OPT. In addition, this methodology offers the advantage of reducing operational and capital costs due to minimizing the process for ethanol production by excluding expensive processes related to detoxification prior to enzymatic hydrolysis and fermentation such as washing/conditioning and solid–liquid separation of pretreated slurry. The potential future use of xylose-digestible microorganisms could further increase the ethanol yield from the proposed process, thereby increasing its effectiveness for the conversion of OPT into biofuels and biochemicals. Recently, lignocellulosic biomass has become recognized for its potential as a renewable natural resource for producing biofuels and biochemicals due to rising interest in exploring alternatives to fossil fuels and reducing global warming. Among the promising lignocellulosic biomass resources, oil palm trunk (OPT) has been spotlighted as a valuable energy resource due to the increased production of palm oil. The palm oil production in from 2012 to 2013 was 28.5 million tons in Indonesia and 18.5 million tons in Malaysia, which amounts to approximately 88 % of the worldwide palm oil production. Moreover, palm oil production is expected to continue growing as the biodiesel industry expands due to the rising demand for palm oil as the raw material for biodiesel . RecentlOPT, unlike general woody biomass, has distinct compositional characteristics. The flesh of OPT contains sap, which accounts for up to 80 % of the felled OPT, depending on its parts. The sap characteristically consists of diverse free sugars, among which glucose is dominant, followed in order by sucrose and fructose, and also small amounts of other sugars . Amino aBesides starch, dried OPT also consists of cell wall components such as cellulose, hemicelluloses, and lignin. However, the structural components of OPT, unlike those of palm trunk sap, cannot be easily converted by biochemical processes due to biomass recalcitrance. Therefore, disrupting the biomass recalcitrance is a prerequisite for converting OPT into the desired chemicals. Hence, a number of pretreatment technologies capable of breaking down the biomass recalcitrance have been developed, which makes cellulose more accessible to cellulolytic enzymes. Among the various pretreatment methods, hydrothermal treatment, also called liquid hot water or autohydrolysis, requires lower capital and operational expenses due to the use of only water as the reaction medium without any chemicals such as acids or bases. Moreover, hydrothermal treatment can reduce the unit process related to the chemical recovery and conditioning for subsequent enzymatic hydrolysis thanks to the rare generation of inhibitory products such as furfural, 5-hydroxymethyl-2-furaldehyde (HMF), and phenolics during the process . To datew/w), on the contrary, it has been reported that further degradation of starch to HMF occurs above 150 °C within a few minutes [Due to the use of only water as the reaction medium, without the need to add any acid or base catalysts, particularly, hydrothermal treatment is suitable for pretreating some starch-rich lignocellulosic biomasses such as OPT since most of the starch can be solubilized under less severe conditions such as autoclaving at 121 °C for 15 min while minimizing its further degradation . In an aFor effective conversion of starch and cellulose in the dried OPT into ethanol, a newly designed and simple method is therefore proposed to minimize starch loss during biochemical conversion and to maximize the production of ethanol and fermentable sugars from OPT. This study also evaluated how much more ethanol production from OPT was increased through the newly designed process than that via conventional process in which OPT was applied to hydrothermal treatment, followed by enzymatic hydrolysis and ethanol fermentation of pretreated OPT slurry.Dami) were kindly supplied by the Korindo Group . The feedstock was ground using a knife mill equipped with a 20-mesh aperture screen, and the powder containing 5.4 % moisture was stored at −20 °C in a refrigerator in sealed plastic bags until use. Sulfuric acid ) as reagent for compositional analysis was purchased commercially. Cellic® CTec2 as a cellulase complex and Cellic® HTec2 as xylanase were purchased from Novozymes Korea , and Novozyme-188 as a supplement to enhance cellobiase activity was purchased from Sigma-Aldrich . Glucoamylase and α-amylase were purchased from Sigma-Aldrich  for enzymatic hydrolysis of starch. Enzyme activities used in this study are described in Table The dried OPT chips  standard procedures [The starch content was determined after converting starch to glucose via enzymatic hydrolysis by amylolytic enzyme mixtures consisting of glucoamylase and α-amylase with a volumetric ratio of 9 to 1. To solubilize starch, 120 g of dry OPT was loaded into a 3-L-scale batch-type fermenter  and then filled with deionized water to a total weight of 1500 g. This soaked slurry was autoclaved at 121 °C for 20 min and cooled down at 50 °C, after which amylolytic enzymes corresponding to 5 μL per 1 g dry biomass were added. Enzymatic hydrolysis of starch was conducted at 50 °C for 24 h with stirring. The liberated glucose was quantified using high-performance liquid chromatography (HPLC) equipped with an autosampler ), a refractive index detector ), and a binary HPLC pump ). An Aminex HPX-87H column  operating at 50 °C was used, and the mobile phase was 5 mM Hocedures , 17. TheEthanol was produced from OPT by two types ethanol conversion processes, which were evaluated in terms of the yields of fermentable sugar and ethanol. As shown in Fig. w/w). Enzymatic hydrolysis of starch was carried out at 50 °C with a stirring speed of 200 rpm for 24 h. Seed culture of Saccharomyces cerevisiae (24858 ATCC) was carried out in YPD medium consisting of 5 g/L yeast extract, 10 g/L peptone, and 25 g/L glucose at 30 °C in a rotary shaker at 200 rpm. After 24-h enzymatic hydrolysis, 5 % (v/v) yeast inoculum was added into enzymatic hydrolysates. No additional nutrients were added to the hydrolysates except on seed culture medium. The fermentation was carried out at 30 °C with a stirring speed of 200 rpm for 24 h. The pH was kept at 5 by adding 15 % ammonia solution. After completion of starch hydrolysis and ethanol fermentation, each aliquot of 1 mL was scooped from well-mixed slurries and separated by centrifugation. Each of the glucose and ethanol in the supernatants was determined using HPLC. The pre-hydrolyzed and fermented PTS (pre-HFPTS) was sequentially used as a material for EECP.Starch in OPT was preferentially converted to ethanol through enzymatic hydrolysis and subsequent ethanol fermentation prior to hydrothermal treatment according to the following detailed method. The OPT equivalent to 120 g of dry biomass loaded in a 3-L-scale batch-type fermenter  was filled with deionized water to a total weight of 800 g and then it was autoclaved at 121 °C for 20 min to solubilize starch from the OPT. After autoclaving, amylolytic enzyme mixtures amounting to 5 μL per 1 g dry biomass were added to the fermenter. These enzyme mixtures were prepared by glucoamylase (4170 U/mL) and α-amylase  with a volumetric ratio of 9 to 1. The slurry was filled with deionized water to a concentration of 10 %  from the PWS-PTS and PWS-HFPTS were referred to as LF-PWS-PTS and LF-PWS-HFPTS, respectively. The 1-mL aliquots of each supernatant were analyzed by HPLC to determine monomeric sugars. To determine liberated monomeric sugars via 4 % acid hydrolysis, in addition, 5-mL aliquots of LF-PWS-PTS and LF-PWS-HFPTS were transferred to pressure vessels  with the addition of 8 % (w/w) sulfuric acid (5 mL) and autoclaved at 121 °C for an hour. The acid-hydrolyzed LFs (AHLFs) from the PWS-PTS and from the PWS-HFPTS were referred to as AHLF-PWS-PTS and AHLF-PWS-HFPTS, respectively.To investigate the effect of solid–liquid separation of PWS on enzymatic digestibility, two kinds of PWS were prepared as follows: auto-PTS and pre-HFPTS, which were subjected to hydrothermal treatment at 180 °C for 30 min using a mini-reactor, respectively. The PWSs from the auto-PTS and pre-HFPTS were referred to as PWS-PTS and PWS-HFPTS, respectively. These PWSs were completely transferred to a 50-mL falcon tube and centrifuged at 3727 × To recover the solid fractions (SFs), remaining PWSs of PWS-PTS and PWS-HFPTS after decanting LFs were filtered with Whatman GF/D glass fiber filter in a 250-mL Buchner funnel with vacuum and washed with 100 mL of deionized water to remove any remaining liquid and pre-fermented ethanol. The recovered SFs from the PWS-PTS and PWS-HFPTS were referred to as SF-PWS-PTS and SF-PWS-HFPTS, respectively. The retained SFs and finely scissored filter were completely transferred to Erlenmeyer flasks (125 mL).w/v) ethanol solution, which was subjected to pretreatment at 180 °C for 30 min, respectively. These PWSs from the starch-free OPT with 5 and 10 % (v/v) ethanol were separated into LFs and SFs, respectively. The recovered SFs were termed SF-PWS-5 % EtOH and SF-PWS-10 % EtOH, respectively, and were subjected to enzymatic hydrolysis.Meanwhile, to investigate the effect of ethanol concentration converted from starch on the hydrothermal treatment performance and resulting glucose yield through enzymatic hydrolysis of cellulose, the starch-free OPT obtained from auto-PTS washed with deionized water was added to a mini-reactor and then filled with 20 mL of 5 and 10 % (w/v) and then autoclaved at 121 °C for 20 min. The auto-PTS was transferred to a reactor vessel and then adjusted with deionized water to a total weight of 1500 g corresponding to a concentration of 8 % (w/v). In EECP, the pre-HFPTS was completely transferred into a reactor vessel as mentioned above. The auto-PTS and pre-HFPTS were subjected to hydrothermal treatment at 180 °C for 30 min. After pretreatment, each slurry was completely transferred into a 3-L-scale batch-type fermenter to which an enzyme mixture of Cellic® CTec2, Cellic® HTec2, and Novozyme-188 (volumetric ratio of 9:1:0.5) corresponding to 0.1 mL per g dry biomass was loaded. Enzymatic hydrolysis was carried out at 50 °C with a stirring speed of 200 rpm for 72 h. The pH was kept at 5 by adding 15 % aqueous ammonia throughout the 72-h hydrolysis. The subsequent ethanol fermentation was performed in the same aforementioned way.Ethanol was produced from OPT by two kinds of laboratory-scale ethanol conversion processes. OPT underwent hydrothermal treatment using a total volume of 2-L-scale stirred Parr reactor . In CECP, the 120 g of OPT (dry biomass basis) loaded in a 1-L media bottle  was filled with deionized water to a concentration of 15 %  were used for enzymatic hydrolysis. The glucose yield from PWS-PTS through enzymatic hydrolysis with enzyme mixtures of Cellic® CTec2 and Cellic® HTec2 was 35.7 g, which was only 66.7 % of TGY. When a certain amount of cellobiase with activity of 554 CBU/g was added to the enzyme mixtures, however, the resulting glucose yield increased to 44.5 g amounting to 83.2 % of TGY, which was increased by around 25 % compared to that without cellobiase addition. Regardless of the addition of cellobiase, on the other hand, glucose yields from PWS-HFPTSs were 24.3 g, which showed a high rate of enzymatic digestibility of cellulose amounting to 91.4 % of TGY for cellulose. These results imply that the oligosaccharides liberated from starch and hemicellulose in PTS could be inhibitory for Cellic® CTec2 during enzymatic hydrolysis. Adding a small amount of cellobiase induced a synergy effect on enzymatic hydrolysis of PWS-PTS with the enzyme mixtures; thus, enhancing glucose yield could be achieved. In addition, it is expected that there are more possibilities for enhancing glucose yield from OPT and overcoming the feedback inhibition, so more research could be needed to find effective enzyme combination. Consequently, converting starch to ethanol prior to hydrothermal treatment facilitates the conversion of as much cellulose as possible to glucose, which is one of alternatives for maximizing ethanol production from OPT by preventing starch from further degradation during the pretreatment.Figure w/w) aqueous ammonia for 8 h, followed by simultaneous enzymatic hydrolysis and fermentation of washed pretreated solid, the final ethanol yield was 10.8 g per 100 g of dry biomass, which corresponds to less than 50 % of TEY [The product yield profiles by CECP and EECP are summarized in Table % of TEY . The lowA total of 100 g dry squeezed OPT was able to be separated into 55 g of PA and 45 g of VB. From the designed process, finally, 11.2 g of ethanol was produced from starch in the PA, which accounted for 85.5 % of TEY from starch. From alkali-pretreated starch-free PA and VB, 5.1 and 8.6 g of ethanol were also obtained, respectively. As a result, a total of 24.9 g of ethanol was generated from the squeezed OPT containing a total available glucan content of 59.8 g, which corresponded to 81.6 % of TEY. This ethanol production strategy using OPT designed by Prawitwong et al. has an advantage of minimizing starch loss but could require an expensive process such as biomass fractionation, chemical use/recovery, and washing/conditioning prior to enzymatic hydrolysis and fermentation [Consequently, EECP could be considered as a suitable process for maximizing ethanol production from lignocellulosic biomass such as OPT that has a lot of starch in the biomass. In addition, EECP has an advantage of reducing operational costs due to the simple ethanol production process that excludes washing/conditioning and solid–liquid separation of pretreated slurry prior to enzymatic hydrolysis. The ethanol fermentation by microorganisms capable of digesting both xylose and glucose could further increase the ethanol yield from EECP. Consequently, EECP would become a more effective strategy for conversion of OPT into biofuels and biochemicals.The available glucose from 100 g dried OPT reached 53.5 g, comprised of 26.9 g of starch as glucose and 26.6 g of cellulose as glucose. Two different processes were applied to the production of ethanol from OPT. The glucose yield from CECP was 43.5 g (81.3 % of TGY), and the resulting ethanol yield was 22.3 g (81.4 % of TEY). In contrast, EECP converted as much starch and cellulose as possible into glucose, i.e., 25.9 g from the starch and 25.6 g from cellulose, which equated to 96.3 % of TGY. The resulting ethanol yield from EECP of 25.6 g (93.5 % of TEY) was 10 % more than that from CECP. These study results confirmed the potential of EECP for use in maximizing ethanol production from OPT through effective starch utilization while minimizing its loss.


Aspergillus terreus. However, the production cost of itaconic acid is too high to be economically competitive with the petrochemical-based products. Itaconic acid is generally produced from raw corn starch, including three steps: enzymatic hydrolysis of corn starch into a glucose-rich syrup by α-amylase and glucoamylase, fermentation, and recovery of itaconic acid. The whole process is very time-consuming and energy-intensive.Itaconic acid is on the DOE (Department of Energy) top 12 list of biotechnologically produced building block chemicals and is produced commercially by A. terreus under the control of the native PcitA promoter. The transformant XH61-5 produced higher itaconate titer from liquefied starch than WT. To further increase the titer by enhancing the secretion capacity of overexpressed glucoamylase, a stronger signal peptide was selected based on the major secreted protein ATEG_02176 (an acid phosphatase precursor) by A. terreus under the itaconate production conditions. Under the control of the stronger signal peptide, the transformant XH86-8 showed higher itaconate production level than XH61-5 from liquefied starch. The itaconate titer was further enhanced through a two-step process involving the vegetative and production phase, and the transformant XH86-8 produced comparable itaconate titer from liquefied starch to current one (~80 g/L) from saccharified starch hydrolysates in industry. The effects of the new signal peptide and the two-step process on itaconate production were investigated and discussed.In order to reduce the production cost, saccharification and fermentation were integrated into one step through overexpressing the glucoamylase gene in A. terreus, which will be helpful for constructing a highly efficient microbial cell factory for itaconate production and for further lowering the production cost of itaconic acid.Itaconic acid could be efficiently produced from liquefied corn starch by overexpressing the glucoamylase gene in The online version of this article (doi:10.1186/s12934-014-0108-1) contains supplementary material, which is available to authorized users. A. terreus [Itaconic acid (methylene succinic acid) is produced commercially by microbial fermentation using the fungus  terreus . It can  terreus ,2. Parti terreus . However terreus .The highest itaconic acid yield is obtained when glucose is used as the substrate, but glucose is too expensive to be used as a raw material for commercial production of itaconic acid. Therefore, other raw materials that are cheaper than glucose, such as starch, molasses, hydrolysates of corn syrup or wood, were also tested. Among them, corn starch is one of the best carbon sources, since it is pure, relatively inexpensive, and stable in a mass supply –6.A. terreus due to gelatinization upon heat sterilization [A. niger follows similar procedures. However, given that A. niger secretes glucoamylase up to > 10 g/l [A. niger, which showed the increased glucoamylase activity [A. niger.Raw corn starch could not be directly used for itaconic acid production by lization . Itaconilization –8. Enzym> 10 g/l , the sacactivity . It seemA. terreus. Instead, Kirimura et al. generated a fusant strain between A. terreus and A. usamii (a glucoamylase producer) producing maximally 35.9 g/L itaconic acid from soluble starch at day 6 of cultivation, whose productivity was five times as high as that of the parental A. terreus strain [A. terreus is genetically engineered to directly produce itaconic acid from liquefied starch through integrating saccharification and fermentation into one step, which would be achieved by overexpressing glucoamylases in A. terreus, thus direct production of itaconic acid from liquefied starch can be realized. To achieve this goal, at least three biological parts are required: an efficient promoter for glucoamylase overexpression in A. terreus, an appropriate glucoamylase gene and an appropriate signal peptide for high-level secretion of overexpressed glucoamylase.Though there are lots of studies on itaconic acid production from starch hydrolysates ,7, theres strain . Howevers strain . ConsideA. terreus is the gpd promoter, which has been successfully applied in driving vgb (the Vitreoscilla hemoglobin gene) overexpression in A. terreus [PgpdA from A. nidulans was by far the most frequently used promoter for genetic modification of A. terreus [A. terreus is expected to be a strong constitutive and native promoter under the itaconic acid production conditions.The constitutive promoters which are not dependent on the carbon or nitrogen source or the specific inducers, and the native ones which are more efficient in directing gene expression than the heterologous ones, are preferred for genetic engineering ,15. So f terreus . PgpdA f terreus ,18. In o terreus ,20. SincA. niger is an excellent glucoamylase producer, and can secrete glucoamylase up to >10 g/l [A. niger has been commercialized and often used for saccharification [It is known that  >10 g/l . Glucoamfication .Since glucoamylases need to be secreted out of the cells for efficient saccharification of liquefied starch, choosing the appropriate signal peptides is essential for high-level secretion of overexpressed glucoamylases .A. terreus was achieved by overexpressing glucoamylase from A. niger under the control of the native citrate synthase promoter, and the itaconate titer was further improved through using the native signal peptide of glucoamylase and optimizing the fermentation conditions. Therefore, an efficient microbial cell factory for direct production of itaconic acid from liquefied starch was established.In the current study, direct production of itaconic acid from liquefied starch by citA gene  of A. terreus CICC 40205 was cloned and sequenced, showing 94.7% sequence identity with the annotated one for ATEG_07990 in the genome sequence of A. terreus NIH 2624. In the upstream of citA from A. terreus CICC 40205, additional twelve nucleotide bases aaaaataaaaag were inserted between −398 and −409 from ATG (A as +1). The 1878-bp upstream sequence of the citA gene has been deposited at EBI (European Bioinformatics Institute) with the accession numberHG530534.A 1878-bp upstream sequence from the start codon (ATG) of the citA gene was evaluated using sgfp  as a reporter [PcitA1  was constructed and transformed into A. terreus CICC 40205 . These results clearly demonstrated that PcitA1 successfully drove sgfp expression in A. terreus CICC 40205.The promoter function of the putative promoter region of the reporter . The expPcitA2 (−1121 bp from the start codon (ATG) of citA, A as +1), PcitA3 (−610 bp from the start codon (ATG) of citA), and PcitA4 (−262 bp from the start codon (ATG) of citA) were designed, among which PcitA4 contains the three conserved stretches found in the putative promoter regions of citA gene from seven Aspergilli [A. terreus CICC 40205 respectively  with its native signal peptide was cloned into pXH43 and pXH44 modified from pXH33 and pXH34 respectively  overexpressed glucoamylase could be effectively secreted out of cells by its native signal peptide; 3) overexpressed glucoamylase was active in saccharification of liquefied corn starch; 4) statistically , PcitA3 is better than PcitA4  the glucoamylase gene A. terreus CICC 40205 under the itaconate production conditions. The major proteins indicated by box  of the secreted proteins by A. terreus CICC 40205. Likewise, introducing the glucoamylase gene with the stronger signal peptide into A. terreus didn’t greatly impact itaconate production while saccharified corn starch hydrolysate was utilized as the carbon source . To further improve the itaconic acid titer, a two-stage process including the vegetative (growing a pre-culture on saccharified corn starch hydrolysates) and fermentation (liquefied corn starch used as the substrate for further fermentation) phase was applied. The parental strain, and the transformants XH61-5 and XH86-8 were directly compared using liquefied corn starch as the carbon source in order to investigate the impact of the two-stage process as well as replacement of the signal peptide of gluoamylase on the performance of three strains, including growth, changes of residual glucose and oligosaccharides, itaconate titer, and glucoamylase production and activity.glaA gene didn’t have serious adverse effects on the growth of A. terreus. All three strains grew faster before 36 hr in the two-step process than in the one-step process respectively, and reached the stationary phase after 48 hr. The growth rates were determined based on these . It appears that WT experienced the stages of carbon starvation and adaptive response to carbon starvation, which might induce complex alterations in the transcriptome, including the expression of genes encoding important elements of primary and secondary metabolism and programmed cell death processes, and complex well-regulated and energy-consuming physiological and morphological changes [Based on the changes of glucose  could directly produce itaconic acid from soluble starch, but the itaconate titer and production rate are too low to be used for industrial production [Citric acid can be directly produced from corn and potato starch by some mutant strains of oamylase ,11. Moreoduction . These rA. terreus from liquefied starch has been achieved through overexpression of the glucoamylase gene glaA in A. terreus under the control of the native gpd promoters. Our results also indicated that the parental strain could directly produce itaconic acid from liquefied corn starch, but the titer was too low compared with the glaA transformants. It has been reported that glucoamylase production from A. terreus could be induced by rice bran, maltose, and cellobiose, and was successfully purified from the culture filtrate of A. terreus [A. terreus was not as high as A. niger, which is not sufficient for direct production of itaconate from liquefied starch by A. terreus.In this study, direct production of itaconic acid by genetically engineered  terreus ,28. HoweA. terreus CICC 40205 under the itaconate production conditions was identified to be an acid phosphatase precursor. Considering that itaconic acid is generally produced under the acidic and phosphate limited conditions [A. terreus CICC 40205 under the optimal itaconate production conditions. However, Han et al. observed that oryzin (ATEG_03900) and ATEG_07481 (a predicted protein) were the most abundant extracellular proteins of A. terreus grown at pH 7.3 on different carbon sources: sucrose, glucose, and starch [The major protein, ATEG_02176, secreted by d starch . ATEG_02d starch ,31.Candida antarctica lipase B with its native signal peptide in Pichia pastoris was achieved [et al. improved protein secretion from Lactococcus lactis through engineering the native signal peptide of a major secreted lactococcal protein, Usp45 [Higher itaconate production level from liquefied corn starch was obtained under the control of the signal peptide of ATEG_02176, and the results of SDS-PAGE, changes of residual glucose and oligosaccharides, and glucoamylase activity assays were consistent with this. The observed results could presumably arise from enhanced secretion of overexpressed glucoamylase by the stronger signal peptide of ATEG_02176. Some examples have demonstrated that protein secretion could be improved through using the strong signal peptides or engineering them. For instance, enhanced secretion of achieved . Ng et an, Usp45 .A. terreus and A. usamii (a glucoamylase producer) [The two-stage process, including the vegetative and fermentation phase, showed higher itaconate production level than the one-stage process, demonstrating that the two-stage process is beneficial for itaconate production, and changes of residual glucose and oligosaccharides, SDS-PAGE and glucoamylase activity assays also supported the conclusion. Under the optimized fermentation conditions, the best genetically engineered itaconate producer XH86-8 produced 77.6 g/L itaconic acid from liquefied corn starch after 3 days of cultivation, very close to the industrial level (~80 g/L) from saccharified starch hydrolysates ,7, and sroducer) .Thus, an efficient microbial cell factory for direct production of itaconic acid from liquefied corn starch has been established. Further improvement of the strain XH86-8 and optimization of the cultivation conditions, including medium compositions, might lead to further enhanced itaconic acid production from liquefied starch.A. terreus was successfully genetically engineered to directly produce itaconic acid from liquefied corn starch through overexpressing the glucoamylases gene. The itaconate titer was increased by replacing the glucoamylase’s native signal peptide with the stronger one. By using a two-step fermentation process, the itaconic acid titer of the best genetically engineered itaconate producer reached 77.6 g/L from liquefied corn starch, which is comparable to the production level from saccharified starch hydrolysates in industry. An efficient microbial cell factory for direct production of itaconic acid from liquefied corn starch has been established, which will be beneficial for biotechnological production of itaconic acid.In conclusion, Taq and pfu DNA polymerases, RevertAid Reverse Transcriptase, restriction endonucleases were from Fermentas  or New England BioLabs . The kits used for molecular cloning were from Omega Bio-tek Biotechnology . Difco™ Potato Dextrose Agar was from BD. The cloning vector pMD18-T-simple was purchased from Takara Biotechnology . Trizol was from Invitrogen Life Technologies Corporation . Hygromycin B was obtained from Solarbio Science Technology Co., Ltd (China). ReadyStrip™ IPG Strips and Bio-Lyte 3/10 Ampholyte were from Bio-Rad . Corn starch was purchased from a local flour mill . α-Amylase (SUHONG AA Plus) and glucoamylase (SUHONG GA II) were obtained from Suzhou Hongda Enzyme Co., Ltd (China).Chemicals and trypsin were from Sigma , Merck  or Ameresco . Oligonucleotides were synthesized by Shanghai Sangon Biotech Co. Ltd (China). Liquefied corn starch was prepared following the recommendation of the enzyme manufacturer. Calcium chloride  was dissolved in hot water at 60°C, then corn starch was added at 1 to 1 ratio (w/v), and pH was adjusted to 6.0. α-Amylase  was supplied, and cornstarch suspension was subsequently sprayed at 106-108°C for 1–2 min, then maintained at 97-98°C for about 1.5 hr  value of liquefied corn starch is around 20). The slurry was then cooled to 60-62°C, and pH was adjusted to pH 4.0. Finally, glucoamylase  was added, and hydrolysis was carried out at 60-62°C for 27–28 hr.Escherichia coli DH5α was used for routine DNA transformation and plasmid isolation, and grown in Luria-Bertani broth at 37°C. A. terreus CICC 40205 from China Centre of Industrial Culture Collection was used in this study. Spores were harvested from 7-day-old potato dextrose agar plate (PDA). Cultivation in shake flasks was carried out in 500 ml non-baffled shake flask containing 55 ml itaconic acid production medium (IPM) on a rotary shaker at 200 rpm and 37°C [The plasmid pSGF957 was kindly provided by Professor Kim from Seoul National University . Escheriand 37°C .General molecular biology techniques were carried out following the standard procedures .citA gene (ATEG_07990)  was amplified using genomic DNA of A. terreus CICC 40205 as the template and the primers PcitA-F1/PcitA-R , PcitA3 (610 bp) and PcitA4 (262 bp) were amplified from PcitA1using the primers PcitA-F2/PcitA-R, PcitA-F3/PcitA-R, and PcitA-F4/PcitA-R respectively. The candidate promoters were cloned into the vector pXH2-1 at the restrictions sites of XhoI and BstBI  were constructed separately. The direction of inserted TtrpC was confirmed by PCR using primers PcitA-F4/TtrpC-R. The signal peptide of glucoamylase was predicted by SignalP 3.0 .The glucoamylase gene Bright field and fluorescent images of the selected transformants at the stage of conidia, young hyphae, and mature hyphae, were taken on fluorescence microscope (Olympus BX51). Young and mature hyphae were obtained by cultivation in shake flasks at 37°C for 11 hr and 36 hr respectively.The fluorescence intensity of two randomly selected transformants for each promoter was determined according to a published procedure .A. terreus CICC 40205 were prepared as previously described by Han et al. [http://www.ncbi.nlm.nih.gov/) was used to identify the target protein. The signal peptide of the identified protein was predicted using SignalP 3.0.The extracellular proteins of n et al. , and anaglaA without its own signal peptide was amplified by PCR using the primers glaA-F2 and glaA-R and cloned into pXH84at the restriction sites of SpeI and BglII to obtain pXH85  named as SP02176-glaA1 was amplified from pXH85 by the primers M13-47 and M13-48, and cloned into the vector pXH43 at the restriction sites of BstBI and BclI (BglII) to get the expression cassette pXH86 containing SP02176-glaA1  as the starting material on a rotary shaker at 37°C for 72 h. The shaking diameter of the rotary shaker is 9 cm. The better itaconate producers were further screened using liquefied corn starch (corresponding to 140 g/L glucose) as the carbon source, which was prepared according to the published procedure [2SO4 at a flow rate of 0.6 ml/min. Authentic itaconic acid was used as a standard to calculate the final concentration of itaconic acid.The titer of itaconic acid produced was determined by HPLC (High Performance Liquid Chromatography) using an Aminex HPX-87-H column (300 mm × 7.8 mm) detected at 210 nm. The column was operated at 35°C with a mobile phase of 4 mM HThe evaporation effects were taken into account when determining the itaconate and other concentrations. To maintain humidity and minimize water evaporation, water was sprayed regularly.7 Spores were first inoculated in 55 ml IPM  with 500 ml shake flasks for 16 hr at 37°C, 200 rpm, and 5 ml seed culture was then transferred into 50 ml IPM , and cultivated for 72 hr at 37°C, 200 rpm.The optimized fermentation was carried out in a two-step process, involving the vegetative (growing a pre-culture on saccharified corn starch hydrolysates) and production phase (liquefied corn starch used as the carbon source for further fermentation) . 2.5 × 1WT and the transformants XH61-5 and XH86-8 were directly compared in the one-step and two-step processes using liquefied corn starch  as the carbon source. Three independents experiments were set for each strain. All assays were done at least in duplicate.Mycelial dry weight was determined as described previously .The activity of overexpressed glucoamylase was assayed following a standard procedure . One uniA. terreus following the protocol [SDS-PAGE was directly performed for the culture filtrate of protocol .Residual glucose was quantified using the biosensor SBA-40C) from Biology Institute of Shandong Academy of Science . Residual oligosaccharides were quantified following a published procedure with slight modification (only glucoamylase was used) [C from Bi