Pretreatment Medium Research Articles

Cryopreservation of Date Palm Pro-Embryonic Masses Using the D Cryo-plate Technique.

In this chapter, we describe a cryopreservation (liquid nitrogen, -196°C) protocol developed for long-term storage of date palm pro-embryonic masses (PEMs), which uses the recently established D cryo-plate technique. Clumps of PEMs (3-5mm in size) were dissected from PEM cultures and placed on pretreatment medium containing 171g/L sucrose for 3days. Clumps were placed in the wells of aluminum cryo-plates in which they were made to adhere using droplets of 3% calcium alginate. PEMs were treated for 20min with a loading solution containing 184g/L glycerol and 136.8g/L sucrose. They were then dehydrated for 90-120min in the air current of a laminar airflow cabinet and immersed directly in liquid nitrogen. For rewarming, the cryo-plates holding the PEMs were immersed for 15min in an unloading solution containing 410.4g/L sucrose. The PEMs were then detached from the cryo-plates, placed for 3days in the dark on posttreatment medium containing 102.6g/L sucrose, and transferred on recovery medium under light conditions. Using this protocol, 74.6 and 95.8% recovery were achieved with the PEMs of the two cultivars tested, Sukkari and Sultany.

Isolation and Characterization of Organosolv and Alkaline Lignins from Hardwood and Softwood Biomass

Isolation of lignins from hardwood and softwood biomass samples, containing 26.1% and 28.1% lignin, respectively, has been performed with the use of alkaline and organosolv pretreatment methods. The effect of catalyst loading, ethanol content, particle size, and pretreatment time on the yields and properties of the isolated lignins were investigated. Alkaline lignins had higher carbohydrate content—up to 30%—and exhibited higher molecular weights in the range of 3000 Da, with a maximum phenolic hydroxyl content of 1 mmol g–1 for birch and 2 mmol g–1 for spruce. Organosolv lignins, on the other hand, showed high purity—93% or higher—despite the more extensive biomass dissolution into the pretreatment medium; they also exhibited a lower range of molecular weights between 600 and 1600 Da depending on the source and pretreatment conditions. Due to the lower molecular weight, phenolic hydroxyl content was also increased, reaching as high as 4 mmol g–1 with a simultaneous decrease in aliphatic hydroxyl content ...

Effect of Genotype, Stress Pre-Treatment and Culture Medium on plant Regeneration of Barley Microspores Via Anther Culture

Effect of Genotype, Stress Pre-Treatment and Culture Medium on plant Regeneration of Barley Microspores Via Anther Culture

Effect of Alkaline Peroxide Pre-treatment on Microfibrillated Cellulose from Oil Palm Fronds Rachis Amenable for Pulp and Paper and Bio-composite Production

Effects of alkaline peroxide (AP) pre-treatment were investigated with respect to the extracted cellulose fibres from the vascular bundles of oil palm (Elaeis guineensis) fronds (OPF) rachis at different AP concentrations. The extracted fibres were prepared through the mechanical fibrillation resulting from the AP pre-treatment concentrations of the rachis. The cellulose fibres obtained were characterized using microscopic (SEM), spectroscopic (FTIR), thermal (TGA-DTG), and X-ray diffraction (XRD) techniques. The screen pulp yield was between 38.07% and 42.69%, which increased with the increase in the AP concentrations. The SEM showed a significant separation of the fibres after the AP pretreatment. FTIR spectroscopy and TGA showed significant dissolution of both lignin and hemicellulose molecules from the treated biomass at higher alkaline peroxide concentrations. The thermal stability of the extracted fibres ranged from 366 oC to 392 oC while the XRD results showed that the cellulose fibre extracted at H2O2/NaOH ratio of 2.5%: 2.0%,w/v AP concentrations gave the highest percentage crystallinity (35.7%). The handsheet made from the cellulose fibre showed that tensile, burst, and tear indexes increased with an increase in AP concentration. Duncan Multiple Range Test shows that mild alkaline peroxide pretreatment (medium concentrations) is best favoured for paper making pulp and bio-composite production.

Enhancement in androgenesis efficiency in barley (Hordeum vulgare L.) and bread wheat (Triticum aestivum L.) by the addition of dimethyl sulfoxide to the mannitol pretreatment medium

Dimethyl sulfoxide (DMSO) is a well-known solvent widely used in cell biology owing to its specific physicochemical properties, which allow it to decrease the lipid bilayer’s thickness and membrane fluidity while increasing membrane permeability. To improve doubled haploid production using an anther culture method in barley and bread wheat, the addition of DMSO to the pretreatment medium was tested. The first experiment was carried out on four barley cultivars with varying degrees of androgenetic ability by exposing them to four DMSO concentrations (0, 0.2, 1 and 2 % v/v). The medium with 1 % DMSO was the most effective in increasing the numbers of embryos and plants. The highest concentration tested (2 %) negatively impacted all of the measured variables when compared with the results of the 1 % DMSO addition, probably owing to a toxic effect. The effects caused by this solvent were more remarkable on the most recalcitrant cultivars, in which there was a threefold increase in the number of green plants. Furthermore, a downward trend in the albinism rate was observed as the concentration of DMSO increased. In a second experiment, we compared a 1 % DMSO supplement with the control to determine whether its addition was effective in several cultivars and F1 crosses of bread wheat. As in barley, there was a marked increase in the number of green plants, leading to a twofold to fourfold increase in both cultivars and F1 crosses.

Microwave assisted chemical pretreatment of Miscanthus under different temperature regimes

Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation biofuels. The most efficient and realistic method to produce fermentable sugars from lignocellulosic biomass is by enzymatic hydrolysis, assisted by thermo-chemical pretreatment. Recently, microwave technology has drawn growing attention, because of its unique effects and performance on biomass. In this work, microwave energy was applied to facilitate NaOH and H2SO4 pretreatment for Miscanthus under different temperatures (130–200 °C) for 20 min. The yields of reducing sugars from Miscanthus during the pretreatment process increased up to 180 °C and then declined with increasing temperature. Out results here showed a remarkable sugar yield from available carbohydrate (73 %) at the temperature of 180 °C by using 0.2 M H2SO4. In comparison with conventional heating pretreatment studied at same temperature with same biomass material, the reducing sugar release in this study was 17 times higher within half the time. It was highlighted that the major sugar component could be tuned by changing pretreatment temperature or pretreatment media. Optimally, the glucose and xylose yield from available carbohydrate are 47 and 22 % by using 0.2 M H2SO4 and NaOH respectively when temperature was 180 °C. The digestibility of pretreated Miscanthus was 10 times higher than that of untreated biomass. 68–86 % of the lignin content was removed from biomass by 0.2 M NaOH. Simultaneous saccharification fermentation (SSF) results showed an ethanol production of 143–152 mg/g biomass by using H2SO4/NaOH microwave assisted pretreatment, which is 7 times higher than that of untreated Miscanthus. Biomass morphology was studied by SEM, showing temperature has a strong influence on lignin removal process, as different lignin deposits were observed. At the temperature of 180 °C, NaOH pretreated biomass presented highly exposed fibres, which is a very important biomass characteristic for improved enzymatic hydrolysis. Compared to conventional pretreatment, microwave assisted pretreatment is more energy efficient and faster, due to its unique heating mechanism leading to direct interaction between the polar part of biomass and electromagnetic field. The results of this work present promising potential for using microwave to assist biomass thermo-chemical pretreatment.

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.

Texture analysis as imaging biomarker of tumoral response to neoadjuvant chemoradiotherapy in rectal cancer patients studied with 3-T magnetic resonance.

The aim of this study was to determine whether texture features of rectal cancer on T2-weighted (T2w) magnetic resonance images can predict tumoral response in patients treated with neoadjuvant chemoradiotherapy (CRT). We prospectively enrolled 15 consecutive patients (6 women, 63.2 ± 13.4 years) with rectal cancer, who underwent pretreatment and midtreatment 3-T magnetic resonance imaging. Treatment protocol consisted of neoadjuvant CRT with oxaliplatin and 5-fluorouracile. Texture analysis using a filtration-histogram technique was performed using a commercial research software algorithm (TexRAD Ltd, Somerset, England, United Kingdom) on unenhanced axial T2w images by manually delineating a region of interest around the tumor outline for the largest cross-sectional area. The technique selectively filters and extracts textures at different anatomic scales followed by quantification of the histogram using kurtosis, entropy, skewness, and mean value of positive pixels. After CRT, all patients underwent complete surgical resection and the surgical specimen served as the gold standard. Six patients showed pathological complete response (pCR), and 4 patients, partial response (PR). Five patients were classified as nonresponders (NRs). Pretreatment medium texture-scale quantified as kurtosis was significantly lower in the pCR subgroup in comparison with the PR + NR subgroup (P = 0.01). Midtreatment kurtosis without filtration was significantly higher in pCR in comparison with PR + NR (P = 0.045). The change in kurtosis between midtreatment and pretreatment images was significantly lower in the PR + NR subgroup compared with the pCR subgroup (P = 0.038). Pretreatment area under the receiver operating characteristic curves, to discriminate between pCR and PR + NR, was significantly higher for kurtosis (0.907, P < 0.001) compared with all other parameters. The optimal cutoff value for pretreatment kurtosis was 0.19 or less. Using this value, the sensitivity and specificity for pCR prediction were 100% and 77.8%, respectively. Texture parameters derived from T2w images of rectal cancer have the potential to act as imaging biomarkers of tumoral response to neoadjuvant CRT.

Production of nano bacterial cellulose from waste water of candied jujube-processing industry using Acetobacter xylinum.

The work is aimed to investigate the suitability of waste water of candied jujube-processing industry for the production of bacterial cellulose (BC) by Gluconacetobacter xylinum CGMCC No.2955 and to study the structure properties of bacterial cellulose membranes. After acid pretreatment, the glucose of hydrolysate was higher than that of waste water of candied jujube. The volumetric yield of bacterial cellulose in hydrolysate was 2.25 g/L, which was 1.5-folds of that in waste water of candied jujube. The structures indicated that the fiber size distribution was 3-14 nm in those media with an average diameter being around 5.9 nm. The crystallinity index of BC from pretreatment medium was lower than that of without pretreatment medium and BCs from various media had similar chemical binding. Ammonium citrate was a key factor for improving production yield and the crystallinity index of BC.

Pilot scale study on steam explosion and mass balance for higher sugar recovery from rice straw

Pretreatment of rice straw on pilot scale steam explosion has been attempted to achieve maximum sugar recovery. Three different reaction media viz. water, sulfuric acid and phosphoric acid (0.5%, w/w) were explored for pretreatment by varying operating temperature (160, 180 and 200°C) and reaction time (5 and 10min). Using water and 0.5% SA showed almost similar sugar recovery (∼87%) at 200 and 180°C respectively. However, detailed studies showed that the former caused higher production of oligomeric sugars (13.56g/L) than the later (3.34g/L). Monomeric sugar, followed the reverse trend (7.83 and 11.62g/L respectively). Higher oligomers have a pronounced effect in reducing enzymatic sugar yield as observed in case of water. Mass balance studies for water and SA assisted SE gave total saccharification yield as 81.8% and 77.1% respectively. However, techno-economical viability will have a trade-off between these advantages and disadvantages offered by the pretreatment medium.

Fast pyrolysis processing of surfactant washed Miscanthus

Miscanthus×giganteus was subjected to pre-treatment with deionised water, hydrochloric acid or Triton X-100 surfactant, and subsequently fast pyrolysed in a fluidised bed reactor at 535°C to obtain bio-oil. Triton X-100 surfactant was identified as a promising pre-treatment medium for removal of inorganic matter because its physicochemical nature was expected to mobilise inorganic matter in the biomass matrix. The influence of different concentrations of Triton X-100 pre-treatment solutions on the quality of bio-oil produced from fast pyrolysis was studied, as defined by a single phase bio-oil, viscosity index and water content index. The highest concentration of Triton X-100 surfactant produced the best quality bio-oil with high organic yield and low reaction water content. The calculated viscosity index from the accelerated ageing test showed that bio-oil stability improved as the concentration of Triton X-100 increased.

Extraction optimization and bioactivities of an extracellular polysaccharide produced by Aspergillus fumigatus

Aspergillus fumigatus produces an extracellular polysaccharide, AFEPS, when grew in NaOH solution pretreated rice-straw medium. A three-level, three-factor Box–Behnken design (BBD) response surface methodology (RSM) was applied to optimize the extraction parameters of AFEPS. RSM analysis indicated good correspondence between experimental and predicted values. The optimal conditions for polysaccharide were: precipitation time 10.9h, pH 5.2 and ethanol concentration 90%. AFEPS was composed of arabinose, xylose and glucose in a molar ratio of 1.05:5.36:10.83, its average molecular weight was estimated to be about 36.2kDa. Evaluation of the antioxidant activity in vitro suggested that AFEPS had high scavenging activity for superoxide anion and hydroxyl radicals. AFEPS exhibited excellent antitumor activities both in vitro and in vivo while showing no damage to normal cells.

Enhanced enzymatic cellulose hydrolysis by subcritical carbon dioxide pretreatment of sugarcane bagasse

Most biomass pretreatment processes for sugar production are run at low-solid concentration (<10 wt.%). Subcritical carbon dioxide (CO2) could provide a more sustainable pretreatment medium while using relative high-solid contents (15 wt.%). The effects of subcritical CO2 pretreatment of sugarcane bagasse to the solid and glucan recoveries at different pretreatment conditions were investigated. Subsequently, enzymatic hydrolysis at different hydrolysis time was applied to obtain maximal glucose yield, which can be used for ethanol fermentation. The maximum glucose yield in enzyme hydrolyzate reached 38.5 g based on 100g raw material after 72 h of enzymatic hydrolysis, representing 93.0% glucose in sugarcane bagasse. The enhanced digestibilities of subcritical CO2 pretreated sugarcane bagasse were due to the removal of hemicellulose, which were confirmed by XRD, FTIR, SEM, and TGA analyses.

Carbon Dioxide Torrefaction of Woody Biomass

The major drawback of biomass for direct combustion applications is its lower heating values and poor grindability when compared to conventional fossil fuels. Torrefaction is one of the thermochemical pretreatment techniques used to improve the properties of biomass. An inert environment is maintained to prevent oxidation of biomass during torrefaction. A novel method for utilization of carbon dioxide as the pretreatment medium for woody biomass has been investigated in the current study. Previous studies on smaller samples using thermogravimetric analysis (TGA) showed an increased mass loss in the CO2 environment, which was attributed to possible structural changes in the biomass and potential effect of ash constituents in the biomass. However, those claims were not validated. The current study on bigger batches of samples also resulted in increased mass loss when using CO2 compared to using N2 as the torrefaction medium. Scanning electron microscopy (SEM) studies and Brunauer–Emmett–Teller (BET) surface area investigation on the torrefied samples from different environments showed increased internal surface area indicating a mild effect of the CO2 reacting with the samples at temperatures normally employed for torrefaction conditions (200–300 °C). Further, grindability studies were performed on the samples pretreated in CO2 and torrefied in N2. The results on grindability showed improved grindability on using CO2 as the pretreatment medium. Proximate and heating value analysis on the pretreated samples showed an increasing trend in the heating value of the samples with increased temperature. Comparable mass loss at lower temparatures improved grindability and improved fuel properties, makes utilization of carbon dioxide as a torrefaction medium for pretreating biomass for combustion applications an attractive technology.

Effect of high pressure thermal pretreatment on Chlorella vulgaris biomass: Organic matter solubilisation and biochemical methane potential

This study investigates the effect of high pressure thermal hydrolysis on organic matter solubilisation and biogas production from Chlorella vulgaris biomass. Microalgae biomass was subjected to three temperatures, namely 140, 160, and 180°C and two heating times (10 and 20min). Results showed that carbohydrates release prevailed over proteins. Carbohydrates were solubilised concomitantly with increasing temperatures. According to the infrared spectra and monomeric sugars determined in the pretreated medium, temperatures applied clearly affected the solubilisation of structural carbohydrates of the microalgae cell wall. Likewise, thermal pretreatment provided enhanced methane production with regard to the raw algal biomass. Enhanced hydrolysis rate constant supported faster biogas production. Regardless the heating time employed, increasing temperatures depicted increasing methane production. Even thought, organic matter solubilisation was greater at 180°C, the anaerobic biodegradability did not show the same trend. This fact was ascribed to the formation of reaction products that hampered methane production. Best case scenario was achieved by subjecting biomass to 160°C which resulted in 64% methane yield enhancement.

Impact of genotype, plant growth regulators and activated charcoal on embryogenesis induction in microspore culture of pepper (Capsicum annuum L.)

The production of double haploids through androgenesis is used by breeders to produce homozygous lines in a single generation. Androgenesis can be achieved by isolated microspore culture, which, however, allows the production of embryogenesis with a very low efficiency. In order to improve the overall embryogenesis in pepper, we study the differences of microspore embryogenesis in different genotypes of pepper, and also document the effect of growth regulators in pretreatment media, and activated charcoal (AC) on embryogenesis induction. Fifty different pepper genotypes were evaluated, and the swollen rate of microspores from different genotypes varied from 3.11% to 29.56% with the mean value of 13.13%. Microspores from genotype ‘36’ had the highest swollen rate, and the lowest swollen rate of microspores was observed in genotype ‘26’. It was concluded from the statistical results of L9 (33) orthogonal test that changes in the level of BA influenced the swollen rate of microspores more significantly, and the combination of 0mg∙l−1 6-benzyladenine (BA), 0.2mg∙l−1 α-naphthaleneacetic acid (NAA) and 0.5mg∙l−1 kinetinin (Kin) was best. AC at a concentration of 0.05% could act as a promoter of embryogenesis in the microspore culture of different pepper genotypes, while the more significant effect was observed with the low responsive genotypes.

Pretreatment of Cocoa Waste for Bioethanol Production Using Ionic Liquid

One of the major advantages of biofuel over fossil fuel is that it is environmentally friendly but unfortunately most of the chemicals used in the pretreatment of lignocelluloses biomass to produce biofuel can cause adverse effects to the environment. In this study, ionic liquid was used for the pretreatment of cocoa waste. Its effectiveness in the treatment process was compared to the alkalis and acids used in the conventional pretreatment media. The effectiveness of pretreatment using ionic liquid, H2SO4 and NaOH was based on the reduction of biomass, production of reducing sugar and also bioethanol. Ionic liquid pretreatment was found to show minimal biomass loss of only 31% after pretreatment compared to H2SO4 and NaOH which showed loss of 61% and 79% respectively. The untreated biomass has 10% amount of cellulose but upon pretreatment with ionic liquid, H2SO4 and NaOH, significant amount of cellulose was detected compared to NaOH which produced only 7% of cellulose. Two types of yeasts were also isolated from Malaysian local fermented food, the tapai ubi which were tested for the abilities to ferment the reducing sugar produced. Using the DNS method for determining reducing sugar, ionic liquid pretreatment was shown to produce 6.3×10–2g/L of reducing sugar while the untreated, H2SO4 and NaOH pretreatment produced 2.87×10–2g/L, 7.4×10–2g/L and 3.37×10–2g/L respectively at the end of 24 hours of incubation. Bioethanol produced during the fermentation was analysed using gas chromatography. Ionic liquid produced a total of 7.885g/L, H2SO4 produced 7.911g/L NaOH produced 6.824g/L and untreated cocoa waste produced 5.116g/L of ethanol at the end of 24 hours incubation.

Optimization of cholesterol oxidase production by Brevibacterium sp. employing response surface methodology

An ultrasound-assisted emulsification as a pretreatment for cholesterol oxidase production by submerge fermentation using Brevibacterium sp. in a batch system was studied. Medium improvement for the production employing response surface methodology (RSM) was optimized in this paper. The concentration of Tween-80, cholesterol and the time of ultrasonic pretreatment medium were further optimized by RSM. Results from RSM showed that the initial concentration of cholesterol, Tween-80 and pretreatment time exerted a significant effect on cholesterol oxidase production, but these factors did not exert a significant effect on cell growth. The improved medium consisted of 4.076 g/L cholesterol, 0.2932‰ (v/v) Tween-80, 22.361 (min) treatment time, and cholesterol oxidase production reached 1.483 U/ml after 36 h culture, which was 83.57% greater than the control medium.

Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger

Date syrup as an economical source of carbohydrates and immobilized Aspergillus niger J4, which was entrapped in calcium alginate pellets, were employed for enhancing the production of citric acid. Maximum production was achieved by pre-treating date syrup with 1.5% tricalcium phosphate to remove heavy metals. The production of citric acid using a pretreated medium was 38.87% higher than an untreated one that consumed sugar. The appropriate presence of nitrogen, phosphate and magnesium appeared to be important in order for citric acid to accumulate. The production of citric acid and the consumed sugar was higher when using 0.1% ammonium nitrate as the best source of nitrogen. The production of citric acid increased significantly when 0.1g/l of KH2PO4 was added to the medium of date syrup. The addition of magnesium sulfate at the rate of 0.20g/l had a stimulating effect on the production of citric acid. Maximum production of citric acid was obtained when calcium chloride was absent. One of the most important benefits of immobilized cells is their ability and stability to produce citric acid under a repeated batch culture. Over four repeated batches, the production of citric acid production was maintained for 24days when each cycle continued for 144h. The results obtained in the repeated batch cultivation using date syrup confirmed that date syrup could be used as a medium for the industrial production of citric acid.

Two‐temperature stage biphasic CO2–H2O pretreatment of lignocellulosic biomass at high solid loadings

Most biomass pretreatment processes for monosaccharide production are run at low-solid concentration (<10 wt%) and use significant amounts of chemical catalysts. Biphasic CO(2) -H(2) O mixtures could provide a more sustainable pretreatment medium while using high-solid contents. Using a stirred reactor for high solids (40 wt%, biomass water mixture) biphasic CO(2)-H(2) O pretreatment of lignocellulosic biomass allowed us to explore the effects of particle size and mixing on mixed hardwood and switchgrass pretreatment. Subsequently, a two-temperature stage pretreatment was introduced. After optimization, a short high-temperature stage at 210°C (16 min for hardwood and 1 min for switchgrass) was followed by a long low-temperature stage at 160°C for 60 min. Glucan to glucose conversion yields of 83% for hardwood and 80% for switchgrass were obtained. Total molar sugar yields of 65% and 55% were obtained for wood and switchgrass, respectively, which consisted of a 10% points improvement over those obtained during our previous study despite a 10-fold increase in particle size. These yields are similar to those obtained with other major pretreatment technologies for wood and within 10% of major technologies for switchgrass despite the absence of chemical catalysts, the use of large particles (0.95 cm) and high solid contents (40 wt%).