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Impact of moisture content on instant catapult steam explosion pretreatment of sweet potato vine Liu, Li-Yang; Qin, Jin-Cheng; Li, Kai; Mehmood, Muhammad A; Liu, Chen-Guang Nov 27, 2017

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Liu et al. Bioresour. Bioprocess.  (2017) 4:49 DOI 10.1186/s40643-017-0179-zRESEARCHImpact of moisture content on instant catapult steam explosion pretreatment of sweet potato vineLi‑Yang Liu2,3, Jin‑Cheng Qin1,3, Kai Li1, Muhammad Aamer Mehmood1,4* and Chen‑Guang Liu1* Abstract Background: Lignocellulose originating from renewable and sustainable biomass is a promising alternative resource to produce biofuel. However, the complex component, especially high moisture content, leads to a higher cost of transportation and processing. The instant catapult steam explosion (ICSE) pretreatment can exploit the intracellular water of lignocellulosic materials and convert into vapors leading towards the breakdown of the feedstock during the explosion process. However, it is necessary to study the impact of moisture content on the pretreatment.Results: The sugar yield of wet feedstock after ICSE pretreatment reached 88.05%, which was higher when compared to dried and untreated biomass. The utilization of wet feedstock decreased the production of inhibitor and improved the carbohydrate content in ICSE‑treated biomass. There occurred a shrinkage of feedstock after drying process and the mechanical breakage upon ICSE pretreatment. Moreover, not all water was converted into vapor to cause break‑age in the lignocellulose.Conclusion: ICSE has shown to be preferably suitable to pretreat wet sweet potato vine with high moisture content, either fresh or soaked biomass that has been dried before. By using these materials, it would have a higher sugar yield and lower inhibitor production after pretreatment. Based on these advantaged aspects of ICSE platform, two potential strategies are proposed to improve the economic and environmental impacts of pretreatment.Keywords: Pretreatment, Moisture content, Instant catapult steam explosion, Sweet potato vine, Enzymatic hydrolysis© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Open Access*Correspondence:  draamer@gcuf.edu.pk; cg.liu@sjtu.edu.cn 1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China4 Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad 38000, PakistanFull list of author information is available at the end of the articleBackgroundNowadays, lignocellulosic biomass offers a promising alternative to produce biofuel owing to its abundance, renewability, and sustainability. However, the recalcitrant nature of biomass requires additional pretreatment steps to make it susceptible to cellulolytic enzymes (Mosier et  al. 2005). Generally, pretreatment efficiency depends on low moisture content and small particle size of the biomass which also would reduce the cost of transpor-tation (Vidal et  al. 2011). However, traditional drying process is an energy intensive process and the solar irra-diance is limited in countries like China as compared to South Asia or Africa; thus, the air-drying process will occupy vast agriculture area preventing in-time crop rotation. So, to keep the crop rotation framework intact, most of lignocellulosic biomass is burnt on the field dur-ing harvesting seasons in northern China, causing atmos-phere pollution and public health hazards (Tan et  al. 2010; Qu et al. 2012). Alternatively, this low-cost biomass may be subjected to efficient pretreatment which do not involve drying step.Fortunately, steam explosion pretreatment in principle requires higher moisture content in lignocellulose bio-mass, which may improve the efficiency of pretreatment. The biomass is usually treated under high pressure for several seconds to minutes to prompt the hydrolysis of Page 2 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 hemicellulose content and then release the pressure with a short time to break the microstructure of lignocellulose (Galbe and Zacchi 2012). Previous researchers adopted steam explosion to pretreat wet corn stalk and found that higher initial moisture content would improve enzymatic hydrolysis of lignocellulose and reduce 20% of steam con-sumption, because water in feedstock presented a buff-ering effect on reaction during steam explosion process (Sui and Chen 2015).However, the traditional steam explosion instrument will produce abundant of inhibitors such as acetic acid, furfural, coumaric acid, and 5-hydroxymethylfurfural (5-HMF). Moreover, the longer pretreatment time will consume more energy (Cullis et al. 2004). To offset these drawbacks, instant Catapult Steam Explosion (ICSE) was invented. The short de-pressure time (0.0825 s) and pretreatment duration (1–5 min) provide ICSE huge mechanical force to destruct the biomass structure and less energy consumption with simple operation than tra-ditional steam explosion (Gong et al. 2012). Previously, it has been demonstrated that ICSE can improve the enzy-matic degradability of lignocellulose with few inhibitors and less energy consumption (Liu et  al. 2014a). ICSE also has positive impacts on the efficiency of subsequent chemical pretreatments involved with organic solvent, acid, and alkali (Liu et al. 2014b).Sweet potato (Ipomoea batatas) belongs to the family Convolvulaceae, containing abundant starch in the root. So far, China being the largest producer of sweet potato in the world has planted over 6.2 million hectares, and produces over 71 million tons of sweet potatoes, which accounts for the 67% of the global production (Fao 2016). The high sugar content and easily cultivated in saline and alkali land of sweet potato allow it to be used as an excellent resource to produce biofuels (Xia et al. 2013). Although significant advancements have been made on the conversion of sweet potato yet the progress made is not up to the desired lev-els. Many sweet potato vines including leaves, stems, and petiole are disposed in the farming field or used as low-value product like animal feed (Tian et al. 2009). Compar-ing with other lignocellulose resources such as corn stover and rice stalk, sweet potato vine contains higher moisture content and extractives (Jibril et al. 1999). These characters may be helpful to study the effects of lignocellulosic mois-ture content on ICSE pretreatment.In present study, sweet potato vines were used to study the effects of moisture content on ICSE pretreatment by investigating the composition, inhibitors production, sugar yield, and thermal stability, for different feedstock including fresh feedstock, naturally dried feedstock, manually dried feedstock, and soaked dried feedstock. It was studied as a simple and practical method to obtain wet lignocellulose for both industry and lab.MethodsBiomass collection and managementFresh sweet potato vines were collected from a field near the city of Dalian in China during the summer, which contained 16.8% cellulose, 9.6% hemicellulose, 42.89% lignin and ash, 17.6% protein, and 1.5% fat after dried. Collected materials were cut into 1- to 2-cm fragments by scissors, and stored at − 20 °C for further use.Fresh feedstocks (FF) were treated in different ways to obtain materials with different moisture contents. FF was dried by natural environment under the sun for 1  week (NDF), which was close to the natural condition before collecting the samples from the field. FF was also dried at 65  °C to the constant weight. This manually dried feed-stock (MDF) is widely accepted for the pretreatment.Samples with coordinating water were also prepared by soaking 20 g MDF into 200 mL of tap water at room temperature at a short time for 2 h (SF-2), medium time (30 h, SF-30), and a long time (60 h, SF-60). To stop the soaking processing and stabilize the moisture content, the Buchner funnels with filter paper were used to sepa-rate the liquid and wet feedstock. Water on the surface of wet feedstock was cleaned by filter paper, following to put in the desiccator to stabilize their moisture con-tent for 24  h. All feedstocks were sealed and stored at − 20 °C.Chemicals and enzymesAnalytical grade glucose, xylose, furfural, 5-hydroxym-ethyl furfural (5-HMF), acetic acid, and coumaric acid were purchased from Sangon Biotech Crop. (Shang-hai, China) and Solarbio Life Science Corp. (Beijing, China). Cellulase (GENENCOR accelerase 1500, cellulase enzyme activity: 105 FPU/mL) was kindly donated by Dupoint Genicor Science Corp. (Shanghai, China).ICSE pretreatment20  g of biomass including FF, NDF, MDF, SF-2, SF-30, and SF-60 were loaded directly into 400-mL chamber of the ICSE equipment (QBS-80B Steam Explosion Test Bed, Henan Hebei, Zhengdao Corp.). Steam with pres-sure (3.25 MPa) and temperature (240 °C) was prepared in advance and sent into the chamber to make the pre-treated pressure be stabilized at 2.8 MPa for 90 s by con-trolling the quantity of flow. After this pretreatment, the ICSE-treated sweet potato vines were released from the chamber by depressurization in 0.0825 s, causing treated material to explode into a stainless-steel cyclone. The slurry (treated material) was collected by scoop into the plastic bag, and then sealed and stored at −  20  °C for further compositional and enzymatic hydrolysis analysis (Liu et al. 2014a).Page 3 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 Moisture content analysisThe moisture content (Cm1) of materials before and after ICSE pretreatment was calculated via the equationwhere X1 is the mass before oven drying and X2 is the mass after drying in the 65  °C oven to get a constant weigh.Inhibitor and sugar analysis after ICSE pretreatment5 grams of each of the ICSE-treated materials were cen-trifuged in 1957×g for 10  min. The supernatants were used for inhibitor and sugar concentration (Ci) analy-sis by high-performance liquid chromatography analy-sis. The solid components were dried at 65  °C to get a constant weight (X3). After the analysis of liquor solu-tion, the inhibitor and sugar yield (mg/g) after ICSE pretreatment were calculated by the equation: inhibi-tor yield = Ci ×  (5 − X3)/X3. All experiments were per-formed in triplicate and the results are presented as mean and standard derivation.Compositional analysisUntreated MDF and solid components of ICSE-pre-treated samples were totally dried and finely ground to less than 40-mesh. 100  mg of each sample was mixed with 1 mL of 72% (w/v) sulfuric acid, and placed in the 50-mL colorimetric tube. The tubes were incubated at 30  °C water bath for 1  h with stirring every 10  min to ensure the intensive mixing. After 1  h, final concen-tration of acid was brought to 4% by adding 28  mL of deionized water, and was put in the autoclave at 121  °C for 1  h. After that, all tubes were left to cool down to room temperature, and the treated samples were filtered by the Buchner funnel with Whatman No.1 filter paper. The supernatants of each tube were used to analyze the glucose, cellobiose, xylose, and arabinose, represent-ing cellulose and hemicellulose, respectively. The solid components were thoroughly washed and dried until it reached a constant weight, representing lignin and ash content. All experiments were performed in triplicate and the results are presented as mean and standard devi-ations (Sluiter et al. 2012).Enzymatic hydrolysisUntreated and the solid fractions of ICSE-pretreated samples were accurately weighted (300 mg) and placed in the 50-mL colorimetric tubes, suspended in 30 mL buffer solution (acetic acid, pH 4.8) containing the enzyme at the ratio of 30  FPU/g. The mixture was incubated at 50 °C for 48 h followed by a centrifugation. Supernatant was subjected to sugar content analysis, as described Cm1 = (X1 − X2)/X1,previously (Liu et al. 2014a, b). The sugar yield was calcu-lated by Eq. (1) below:Glucose and xylose were analyzed by the HPLC. All experiments were performed in triplicate, and results are presented as mean and standard deviations.High‑performance liquid chromatography analysisThe supernatant from ICSE-pretreated samples and enzymatic hydrolysis were filtered through 0.45-μm filter and 20 µL of each sample was loaded to the ion exclusion column (300 mm × 7.8 mm, Bio-Rad, Hercules, Aminex HPX-87H) at 50  °C in HPLC system equipped with a refractive index detector and UV detector (Waters, MA, USA), for the quantitation of inhibitors and saccharides. Sulfuric acid 0.01 mol/L was used as mobile phase at flow rate 0.5 mL/min (Liu et al. 2014a).Scanning electron microscopy (SEM)The microscopic morphology changes in the untreated and ICSE-pretreated samples after dried by lyophiliza-tion were observed using SEM. Each of the samples was placed on the aluminum sample platform and scanned by environmental scanning electron microscopy (Quanta 450, FEI, USA20 kV), using 20 kV as described previously (Liu et al. 2014a).Thermal gravimetric analysis (TGA)About 10 mg of freeze-dried MDF, ICSE-treated FF, and ICSE-treated MDF were placed in the platinum cruci-bles for thermal degradation analyses using TGA Q500 (TA Corporation, USA). Samples were heated from 25 to 500  °C at a constant heating rate of 10  °C/min under nitrogen atmosphere at the flow rate of 50 mL/min (Car-rier et al. 2011).Results and discussionThe change of moisture content in different feedstockMoisture content of any plant biomass is an important parameter for its subsequent usage as a feedstock for bioenergy. To evaluate the effect of moisture content on pretreatment and enzymatic hydrolysis, five forms of samples were prepared (Table  1). FF normally contains 80.50% to sustain the growth of plants. NDF shown to contain 63.57% moisture content which was obtained through exposure of harvested FF under the sun for 7  days. MDF was subjected to soaking to regain the moisture content. Interestingly, the moisture content of SF immediately came up to 62.93% through submerging (1)Sugar yield(%) =[(glucose× 0.9)+(xylose× 0.8)]carbohydrate in biomass× 100.Page 4 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 MDF for 2  h and finally stabilized at around 84.24% at 60  h. The moisture content of SF-60 was close to that of FF, which may be attributed to water absorption by favorable hydrophilic component and the sponge-like structure of lignocellulose (Dhakal et  al. 2007). In addi-tion, it is worth mentioning that, to achieve the same available biomass, the weight of MDF was one-fifth of FF, so the drying process could significantly reduce the total weight of lignocellulose, which is very important for biomass transportation (Axelsson et al. 2012). More-over, soaking is a convenient way to regain the water, if required, after the easy transportation of dried biomass to the biofuel industry.ICSE-pretreated sample contained higher mois-ture content ranging from 93.30 to 97.90%, which was caused by the abundant liquefied water condensed from the steam and stored in the pores of feedstock after the explosion (Table  1). Since the initial moisture content of feedstock also affects the final moisture content, it is obvious to get similar trends of moisture content after pretreatment. However, the ICSE pretreatment demon-strated its capability to smooth the difference of moisture content among all samples, which nullify the impact of drying on ICSE.Composition and inhibitors in pretreated feedstocksThe pretreatment with high temperature that can con-tribute to enhance the available sugars upon hydrolysis is preferred in the bioconversion of biomass to biofuels. However, under high-temperature pretreatment, the lig-nocellulose will be partially degraded to inhibitors such as organic acid, furfural, 5-HMF, which drastically hin-der the efficiency of enzymatic hydrolysis and fermenta-tion process (Jönsson et  al. 2013). The 5-HMF, furfural, and coumaric acid, respectively, represent the cellulose, hemicelluloses, and lignin, and reflect the compositional change of untreated and pretreated biomass (Vander et al. 2014).Table 2 shows that ICSE improved the cellulose content from 16.83% (untreated MDF) to 40.25–48.69% (ICSE treated), accompanied with the reduction of “lignin” from 42.88% (untreated MDF) to 19.66–24.76% (ICSE treated) and the change of hemicelluloses content from 9.60% (untreated MDF) to 8.23–15.95% (ICSE treated). The “lignin” content in the solid phase was higher than that of references’ data, since the protein, fats, and extractives may mix with lignin to impact the acid insoluble lignin and the acid soluble lignin (Jibril et al. 1999). In spite of considerable degradation of hemicelluloses under high temperature, the removal of abundant organic extractives and proteins helped hemicelluloses maintain the content percentage and even better (Zhan et al. 2013; Rocha et al. 2012). This may also explain the significant reduction of “lignin” content in feedstocks after ICSE pretreatment, since extractives, fats, or proteins in detected “lignin” might easily to be hydrolyzed within higher temperature Table 1 The moisture content and the compositional analyses of liquid phase after ICSE pretreatmentData were shown as “mean (standard deviation)”Feedstocks FF NDF MDF SF‑2 SF‑30 SF‑60Moisture content (%) Untreated 80.50 63.57 0.97 62.93 83.38 84.24 Pretreated 96.95 96.04 93.30 95.62 97.90 97.29Liquid contents Pretreated (mg/g)  Glucose 0.7865 (0.0649) 0.3798 (0.0123) 0.2638 (0.0279) 0.0284 (0.0091) 0.0008 (0.0012) 0.0000 (0.0000)  Xylose 0.8858 (0.0577) 0.5393 (0.0292) 0.4733 (0.0320) 0.0682 (0.0137) 0.0031 (0.0020) 0.0006 (0.0000)  Acetic acid 0.0768 (0.0049) 0.0926 (0.0007) 0.1293 (0.0019) 0.0496 (0.0015) 0.0674 (0.0517) 0.0000 (0.0000)  Coumaric acid 0.2405 (0.0827) 0.4769 (0.0303) 0.7838 (0.0149) 0.2797 (0.0136) 0.0659 (0.1110) 0.0241 (0.0000)  5‑HMF 0.0702 (0.0247) 0.0492 (0.0325) 0.0418 (0.0021) 0.0231 (0.0080) 0.0059 (0.0040) 0.0041 (0.0008)  Furfural 24.17 (2.18) 17.41 (0.74) 34.81 (0.96) 18.76 (1.04) 5.37 (1.40) 5.42 (0.24)Table 2 The compositional analysis of untreated MDF and the solid contents after ICSE pretreatedData were shown as “mean (standard deviation)”Cellulose/% Hemicellulose/% Lignin and ash/%Untreated MDF 16.83 (1.86) 9.60 (1.20) 42.88 (0.67)Pretreated FF 40.25 (1.62) 10.27 (0.13) 22.39 (0.52) NDF 41.10 (2.18) 10.92 (0.90) 19.66 (2.91) MDF 41.96 (4.66) 8.23 (1.40) 24.76 (1.86) SF‑2 42.28 (1.75) 10.95 (1.96) 23.88 (4.41) SF‑30 48.69 (1.35) 15.04 (0.92) 20.40 (1.85) SF‑60 48.38 (0.31) 15.95 (0.86) 21.28 (2.77)Page 5 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 during ICSE pretreatment. Figure  1a, b shows the posi-tive correlation for linear fitting between cellulose (hemi-celluloses) and the moisture content in MDF, SF-2, SF-30, and SF-60 after ICSE pretreatment. Those four feedstocks marked in pink color share the oven-drying process in common. The oven-drying process with rapid dehydra-tion of feedstock under higher temperature resulted in significant structure change from FF (Fig. 4). Therefore, it is worth noticing that two cyan scatters representing FF and NDF were non-compliance with the line correlation among those four feedstocks, since the plant’s intracel-lular components would impact the yields of sugar and inhibitor at non-soaking conditions. Figure  1c, d illustrates a negative correlation between glucose (xylose) in the liquid phase and the moisture con-tent. The R2-value was more than 0.98 which strongly validates the reliability of linear relationships. Soaking will dissolve low molecule sugar from feedstock, so the feedstock after long-time soaking lost a plenty of solu-ble sugar. Though the portion of glucose and xylose was derived from the thermal degradation of cellulose and hemicelluloses, ICSE mainly acts as physical pretreat-ment and produces little monosaccharides. Therefore, soaking is the prior factor to reduce the glucose and xylose in liquid phase. Interestingly, FF contained mostly glucose and xylose, which were nutrients for sweet potato cell in the vine after harvesting. After 7 days drying, the glucose and xylose decreased due to continuous respira-tion by living cells in NDF.The inevitable high-temperature process during ICSE pretreatment converts some lignocellulose to inhibitory by-products such as 5-HMF, furfural, acetic acid, and coumaric acid from hexose, pentose, and lignin, respec-tively (Table 1). The correlation of inhibitor and moisture Fig. 1 Effects of moisture content on components, inhibitors, and sugar yield. Carbohydrates (a cellulose, b hemicellulose) in the solid fraction, sugar (c glucose, d xylose), and inhibitors (e acetic acid, f coumaric acid, G 5‑HMF, h furfural) in the liquid fraction, and sugar yield (i) of ICSE‑pre‑treated feedstocks after enzymatic hydrolysis. Cyan plots: FF and NDF; pink plots: MDF, SF‑2, SF‑30, SF‑60Page 6 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 content was consistent with the relation between sugar and moisture content (Fig.  1). Among these inhibitors, the coumaric acid and furfural produced from lignin and hemicelluloses, respectively, were higher than oth-ers, which reflected that ICSE process could more easily hydrolyze lignin and hemicelluloses than cellulose (Liu et al. 2014a). Since soaking is analogous to washing and can rinse monosaccharides and oligosaccharides out of the material surface (Frederick et al. 2014), longer soak-ing time diminished the surface residual sugars and subsequently decreased the accumulation of inhibitors during ICSE pretreatment (Table 1).The pretreatment efficiency is not only determined by the moisture content of feedstock, but also by the method of feedstock handling. Therefore, the structure of feedstock should be analyzed for further understanding of this process.Thermal degradation analysis of untreated and ICSE‑pretreated feedstocksThermal stability of the biomass is analyzed by using TGA, a widely adopted technique to determine the ther-mal degradation of plant biomass (Carrier et  al. 2011; Sanchez-Silva et al. 2012). In general, the differential-TG (DTG) curves of lignocellulose biomass often show three main peaks: one reflects the evaporation of extractives or water at 100–200  °C, the others stand for degrada-tion of cellulose and lignin at 300–350 and 300–500  °C, respectively. The DTG curve usually exhibits shoulders in the temperature range of 200–300  °C, corresponding to the hemicelluloses degradation. The relative intensities of each peak could be used to calculate the quantities of hemicelluloses, cellulose, and lignin present in the ligno-cellulose (Carrier et al. 2011).Interestingly, the respective hemicelluloses shoulder of MDF’s DTG curve almost disappeared after ICSE pretreat-ment and the height of its maximum peak increased 12.5% and moved to a higher temperature due to the degrada-tion of hemicelluloses and the related lifting of cellulose (Fig. 2). Noticeably, it is highly consistent for the tempera-ture and area of DTG curves’ shoulder with a range from 200 to 350  °C between ICSE-FF and ICSE-MDF due to their minor variance of carbohydrate content, less than 0.66%. This finding was further confirmed by the compo-sitional analysis (Table 2). In summary, ICSE pretreatment degraded the hemicelluloses content and nullified the impact of drying process on the change of lignocellulosic composition when compared to wet feedstock.Impact of pretreatment on enzymatic hydrolysisThe enzymatic hydrolysis is the key step to obtain the fermentable sugars from lignocellulosic biomass. Before pretreatment, handling operation enhanced the struc-tural and compositional change of feedstock (Table 2 and Fig. 3), which can be confirmed by sugar yield after enzy-matic hydrolysis. NDF and MDF owned better sugar yield than FF, and soaking duration further improved the sugar yield. The soaking process could wash away some chemi-cal composition that might inhibit enzymolysis or lead to invalid enzymatic adsorption (Frederick et  al. 2014). In addition, the hornification effects due to the drying pro-cess for MDF might lead the plant to have a lower acces-sibility to enzyme or chemical reagents, which might also inhibit the enzymatic hydrolysis (Fernandes et al. 2004).A considerable pretreatment method should ensure that pretreated feedstocks are suitable for robust saccharifica-tion (Agbor et al. 2011). Figure 1 illustrates that ICSE pre-treatment significantly enhanced the enzyme hydrolysis when compared to the untreated feedstocks. Especially, the sugar yield of ICSE-FF was shown a fourfold incre-ment and reached to the maximum value (88.05%) of all samples. At this condition, sugar concentration after enzymatic hydrolysis is about 5.18 g/L. Similar improve-ments were observed in NDF and soaked biomass. These Fig. 2 TGA and DTG analysis of FF, ICSE‑FF, and ICSE‑MDF Fig. 3 Sugar yields of untreated and pretreated feedstockPage 7 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 results indicate that higher moisture content is helpful to improve further enzymatic hydrolysis after ICSE pre-treatment (Figs.  1i and 3); Sui and Chen drew the same conclusion by using normal steam explosion pretreatment on corn stover (Sui and Chen 2015). However, this impact was less obvious on the samples which were soaked for short durations, i.e., 2 h. In addition, whether it is treated or not, the sugar yield of SF-30 was close to SF-60. So, 30  h is enough to improve the degradability of lignocel-lulose. Though the moisture content of NDF and SF-2 was similar (about 63%), their sugar yields were quite different due to the hornification effect. Soaking process enabled lignocellulose to absorb moisture, but the shrinkage of internal fibers could not be fully recovered. It also demon-strated that the moisture content was not the only factor to predominate the enzymatic hydrolysis, but structure of feedstocks might be more pivotal on accessibility of cel-lulase than expected (Fernandes et al. 2004).The morphological and micro‑structural analysis on feedstockSo far, compared with MDF, ICSE pretreatment had more efficient impacts on FF and SF. The macroscopic appear-ance of feedstocks under different conditions is shown in Fig. 4; both FF and SF-60 were full of water, which seem to be more resilient than dried feedstock. Similar with wood industry, the morphological structure of dried wood could get back in shape after absorbing some water (Wang et al. 2006). Feedstock was destructed by steam explosion which turned into more uniform and reduced the parti-cle size. As an order of destruction, FF occupied first place followed by dried feedstocks. It was interesting to note that soaked feedstock produced non-uniform particles when subjected to ICSE and some of the biomass compo-nents were unable to be reached by hot steam. The similar findings were observed previously, because the hornifi-cation effects would lead to different biomass structures between FF and SF and potentially impact ICSE pretreat-ment (Fernandes et al. 2004). Soaking could not infiltrate to all parts due to mass transfer limitation at short time, which formed the un-soaked tissue mainly located in inner part of biomass (Borrega and Kärenlampi 2010).The sample used for SEM micrographs was dried by freeze-drying that preserved cell structure (Fig.  4), whereas the NDF and MDF undergone a drying process at ambient and moderately high temperature, and their cell wall becomes very vulnerable due to dehydration. As shown in Fig. 4, the pores in FF were biggest among three feedstocks, which facilitate the steam or hot water penetrating biomass and enhancing explosion force to Fig. 4 Photographs (round in the left bottom) and SEM microscopic appearance of biomass. The feedstocks included untreated and ICSE‑pre‑treated sweet potato vines for FF, MDF and SF‑60 h. a–c mark the pores of lignocellulosePage 8 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 degrade components. When the feedstock was totally dried, all pores in MDF shrank to about one twenty of area of pores in FF due the hornification effects (Fer-nandes et al. 2004). It blocked the entrance of steam and hot water into pores and lead to a lower accessibility of vapors. If the MDF was soaked in the water, the rehydrat-ing feedstocks tend to return previous status with big pores. Nevertheless, the irreversible ruins on cell wall by drying process have occurred, which leads to the pores of SF much bigger than MDF’s, but still smaller than FF’s.After ICSE pretreatment, all feedstocks were similar because of their similar moisture content (Table  1), but there was still obvious different observations by naked eyes. The particles of ICSE-pretreated FF were the small-est and distributed evenly. Inversely, the particles of ICSE-pretreated MDF were not homogeneous and the particle size was bigger than ICSE-pretreated FF particles. Some big debris could be found in pretreated MDF and SF-60.The role of water and steam in ICSE pretreatmentGiven the basic principle of steam explosion, the mechanical force is generated by the expanding gas (water vapors). So, changes of gas volume during the working temperature range may be helpful to enhance the destruction impact of the steam explosion. For the vaporization from hot water generated huge volume steam subsequently causing biomass degradation.Though the presence of higher moisture content seems attractive for steam explosion, however, the high mois-ture content of biomass would require more energy due to higher specific heat capacity of water. On the other hand, it would not possible for all liquid water to transform into steam during the explosion if the moisture content is high, even with heavier inputs of energy. Roughly calculation for the energy input and steam generation during steam explo-sion can be undertaken as Eq. 4, which showed the climbing amount of steam (ms) with the increase of Cm, where, 1 kg biomass with Cm moisture content was heated from 25 to 230 °C (1.8 MPa). Since the change of specific heat capacity is little within this temperature range, the average values of specific heat capacity for water and feedstock are 4.37 and 1.7 kJ/(kg K), respectively. The vaporization heat of water at 230 °C is 1812.6 kJ/kg. When the Cm is lower than 27.50%, theoretically, the biomass should become completely dry subjected to ICSE. But what happened, the condensed water from heating steam increased the moisture content of pre-treated biomass. Therefore, the moisture content of wet feedstock helped to remove the hemicelluloses and other water-soluble components, and generated more inner steam for improved explosion impact (Sui and Chen 2015).Effects of moisture content on lignocellulose pretreatmentDue to the high moisture content of lignocellulose, researchers had studied its effects on various pretreat-ment methods such as ionic liquid pretreatment, grind-ing, and steam explosion (Table  3). Grinding of wet lignocellulose would consume more energy and finally have a bigger particle size than dried feedstock (Barakat et al. 2015). Mixing water with ionic liquid would lead to a lower pretreatment efficiency, though it would reduce reagents and the whole pretreatment cost (Shi et  al. 2014a).Fortunately, the moisture content of lignocellulose would improve pretreated efficiency of steam explosion or supercritical  CO2 pretreatment. Compared with dried feedstock, wet lignocellulose would prohibit the pyroly-sis of hemicelluloses and facilitates the following enzy-matic hydrolysis during steam explosion pretreatment (Sui and Chen 2015). In addition, previous works about some special steam explosion, such as the pre-soaking lignocellulosic biomass with  SO2 before the pretreat-ment (Cullis et  al. 2004) and the addition of ammonia in the steam water (Moniruzzaman et al. 1997), showed higher sugar yield than ones obtained from dried feed-stock and in agreement with this study. Considering the high cost of lignocellulosic pretreatment, the steam (4)mS =1 kg× (230− 25)K× (1.7 kJ/kg · K× (1− Cm)+ 4.37 kJ/kg · K× Cm)1812.6 kJ/kg.convenient evaluation, if pressure changes are ignored, 1 L air at 25 °C would be 1.7 L at 230 °C (Eq. 2). However, 1 L water at 25 °C would be transformed to 2100 L steam at 230  °C (Eq.  3). Therefore, higher moisture content potentially produces more inner steam for mechanical work which subsequently can destroy the compact struc-ture of biomass. But the steam which is out of feedstock only can convert their thermal energy to kinetic energy.In the small pores of biomass, the steam explosion process followed two steps. The first part is heating: the gas and liquid were heated from room temperature to setting temperature about 230  °C. The water in the bio-mass remained as liquid phase because the working pres-sure was always higher than saturated vapor pressure. So, the liquid hot water dissolved partial lignocelluloses and other water-soluble components. The next step was flash depressurizing. When the pressure in work-ing vessel is instantly released to atmosphere pressure, (2)Air volume =T2P1T1P2V 1 =(230+ 273)K(25+ 273)K× 1L = 1.7L(3)Steam volume =1000g18gmol× 37.8Lmol= 2100LPage 9 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 explosion is a better option. It has an appealing trait, which does not require too much refining operation before pretreatment and allows simplifying the handling on raw feedstock.Though researchers studied moisture effects on pre-treatment efficiency, few of them studied impacts of intracellular moisture. Here, FF showed 88.05% sugar yield after ICSE pretreatment which was competitive when compared to other resources and steam explosion pretreatment. Conclusively, the sweet potato vines and ICSE would be an excellent resource and pretreatment process for the preparation of high-value substrates for microbial fermentation.The strategies for feedstock collection and transportationThe collection, storage, transportation, and pretreat-ment of biomass are energy consuming processes. The moisture content has significant impacts on these pro-cesses (Kudakasseril et  al. 2013). In US, cellulosic etha-nol plant uses common feedstock like agriculture residue, which are naturally dried for 1–2 months in the fields and bunched by rotary baler. The dry feedstock could save the cost of transportation (Axelsson et  al. 2012). However, this path is not suitable for cellulosic plants in China, because the agricultural residues cannot be allowed to stay too long in the field due to the following farming, and the wet feedstock with high moisture content is heavy enough to cost high transportation charges (Shi et al. 2014b).The research has demonstrated that the fresh and soaked biomass (pre-dried) performed better after ICSE pretreatment than dried feedstock, considering the dra-matical improvement of carbohydrate content and sugar yield, and the lower production of inhibitors. Therefore, two strategies based on ICSE pretreatment were pro-posed (Fig. 5) for agricultural countries which are forced to use high-density cultivation such as China and Paki-stan to fulfill the requirements of their huge populations.The first strategy follows the ordinary handling pro-cess before pretreatment, but the dry biomass needs to be soaked to regain the moisture content and to wash out the monosaccharides. The high moisture content will benefit the ICSE performance with the improvement of sugar yield. Attractively the soaked water can also be recycled for next soaking, which concentrates the soluble sugar form raw biomass and avoid the accumulation of inhibitors during thermal pretreatment.In the second proposed strategy, fresh biomass may be directly subjected to ICSE at an adjacent working station near the farm. The liquid phase of pretreated biomass can be either returned to field as fertilizer or utilized for biogas production, since it contains plentiful organic compounds. The second strategy is more suitable to the populous countries, including China, where crop rota-tion is required either due to seasonal issues or due to heavy requirements of food and feed. For example, in eastern China, the crop residue should be moved quickly within 1 month for the following cultivation; thus, there is no time available for field drying (Rasmussen et  al. 1980). Moreover, ICSE can efficiently decrease particle size by destroying the compact structure and elevate the sugar content, when fresh biomass is used. Moreover, Table 3 Summary for the effects of moisture content on lignocellulose pretreatmentNA not availablePretreatment Biomass Sugar yield Advantage Disadvantage Refs.Ionic liquid Switchgrass 70% Decrease the cost of ionic liquid pretreatmentThe existence of water will hamper the efficiency of pretreatmentShi et al. (2014a)Grinding Wheat straw, corn stover etc.NA NA Result in additional energy requirement and higher final particle sizeBarakat et al. (2015)Supercritical  CO2 Southern yellow pine84.7% The increase of initial moisture content will obtain higher final sugar yieldNA Kim and Hong (2001)Steam explosion Corn stover 90% Improve the sugar yield Not benefit for the quickly heating of biomass during pretreatmentSui and Chen (2015)Ammonia fiber steam explosionCorn stalk 80% Increase of moisture content does not hamper the enzymatic hydrolysis of lignocelluloseDitto Moniruzzaman et al. (1997)Steam explosion Softwood 60–90% Prompt the enzymatic hydrolysis and reduce the hydrolysis of hemicelluloses contentDitto Cullis et al. (2004)Acid steam explosion Corn stover > 90%(Xylose)High soluble sugar yield Ditto Emmel et al. (2003)Page 10 of 11Liu et al. Bioresour. Bioprocess.  (2017) 4:49 transportation of the onsite ICSE-pretreated solid to the biofuel producing plant can also save the time and cost, comparing with onsite drying or transporting fresh bio-mass (Fig. 5).ConclusionThe present study focused on developing a low-cost strategy for the pretreatment of feedstock to biofuel-pro-ducing industry. The moisture content of lignocellulosic biomass can be utilized to enhance the enzymatic hydrol-ysis via ICSE, instead of drying. Moreover, ICSE pretreat-ment raises the carbohydrate content up to 1.43-folds with concomitant lowering of the inhibitors in the hydro-lysate. The sugar yield of ICSE-pretreated fresh feedstock was improved by 2.5-folds and reached to 88.05%, which was like soaking the biomass for 60 h, suggesting that the use of fresh biomass would be the best way to run ICSE.AbbreviationsICSE: instant catapult steams explosion; FF: fresh feedstock; NDF: naturally dried feedstock; MDF: manually dried feedstock in oven; SF‑2: soaking feed‑stock for 2 h; SF‑30: soaking feedstock for 30 h; SF‑60: soaking feedstock for 60 h; SEM: scanning electron microscopy; TGA: thermal gravimetric analysis; DTG: differential thermal gravimetric analysis; 5‑HMF: 5‑hydroxymethyl furfural.Authors’ contributionsLL carried out this experiment, data collection, data analysis, and manuscript preparation. CL conducted the research, investigation process, artwork, and manuscript preparation. JQ and KL made equal contribution to prepare initial manuscript. MAM revised the manuscript to its final form. All authors read and approved the final manuscript.Author details1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. 2 Department of Wood Science, University of British Columbia, Vancouver V6T 1Z4, Canada. 3 School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116023, China. 4 Department of Bioinformat‑ics and Biotechnology, Government College University Faisalabad, Faisal‑abad 38000, Pakistan. AcknowledgementsWe appreciate the kind support of Prof. Feng‑Wu Bai. We also would like to thank Xue‑Mi Hao, Bo‑Yu Geng, and Bo‑Wen Jin for technical assistance and valuable discussion.Competing interestsThe authors declare that they have no competing interests.Availability of data and materialsAll data are presented in this main manuscript.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.FundingThis work was supported by the National Natural Science Foundation of China [Grant Numbers 51561145014, 21536006, 21406030].Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub‑lished maps and institutional affiliations.Received: 7 September 2017   Accepted: 14 November 2017ReferencesAgbor VB, Cicek N, Sparling R et al (2011) Biomass pretreatment: fundamentals toward application. Biotechnol Adv 29:675–685. https://doi.org/10.1016/j.biotechadv.2011.05.005Fig. 5 Strategies for collection and pretreatment of biomass before factory production based on ICSEPage 11 of 11Liu et al. 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