Effects of glycerol on enzymatic hydrolysis and ethanol production using sugarcane bagasse pretreated by acidified glycerol solution

In this study, for the first time the effects of glycerol on enzymatic hydrolysis and ethanol fermentation were investigated. Enzymatic hydrolysis was inhibited slightly with 2.0 wt% glycerol, leading to reduction in glucan digestibility from 84.9% without glycerol to 82.9% (72 h). With 5.0 wt% and 10.0 wt% glycerol, glucan digestibility reduced by 4.5% and11.0%, respectively. However, glycerol appeared not detrimental to cellulase enzymes. Ethanol fermentation was not affected with glycerol up to 5.0 wt%, and was inhibited slightly with 10.0 wt% glycerol, which resulted in reduction in ethanol yield from 86.0% without glycerol to 83.7% (20 h). Based on laboratory and pilot scale enzymatic hydrolysis and ethanol production results, it was estimated that 0.142 kg ethanol could be produced from 1.0 kg dry bagasse (a glucan content of 38.0%) after pretreatment with acidified glycerol solution.

The effect of various pH, ionic strength and temperature on papain hydrolysis of salivary film

Stimulated human whole saliva (WS) was used to study the dynamics of papain hydrolysis at defined pH, ionic strength and temperature with the view of reducing an acquired pellicle. A quartz crystal microbalance with dissipation (QCM-D) was used to monitor the changes in frequency due to enzyme hydrolysis of WS films and the hydrolytic parameters were calculated using an empirical model. The morphological and conformational changes of the salivary films before and after enzymatic hydrolysis were characterized by atomic force microscopy (AFM) imaging and grazing angle infrared spectroscopy (GA-FTIR) spectra, respectively. The characteristics of papain hydrolysis of WS films were pH-, ionic strength- and temperature-dependent. The WS films were partially removed by the action of enzyme, resulting thinner and smoother surfaces. The IR data suggested that hydrolysis-induced deformation did not occur onto the remnants salivary films. The processes of papain hydrolysis of WS films can be controlled by properly regulating pH, ionic strength and temperature.

The combination of plant-expressed cellobiohydrolase and low dosages of cellulases for the hydrolysis of sugar cane bagasse

Background The expression of biomass-degrading enzymes (such as cellobiohydrolases) in transgenic plants has the potential to reduce the costs of biomass saccharification by providing a source of enzymes to supplement commercial cellulase mixtures. Cellobiohydrolases are the main enzymes in commercial cellulase mixtures. In the present study, a cellobiohydrolase was expressed in transgenic corn stover leaf and assessed as an additive for two commercial cellulase mixtures for the saccharification of pretreated sugar cane bagasse obtained by different processes. Results Recombinant cellobiohydrolase in the senescent leaves of transgenic corn was extracted using a simple buffer with no concentration step. The extract significantly enhanced the performance of Celluclast 1.5 L (a commercial cellulase mixture) by up to fourfold on sugar cane bagasse pretreated at the pilot scale using a dilute sulfuric acid steam explosion process compared to the commercial cellulase mixture on its own. Also, the extracts were able to enhance the performance of Cellic CTec2 (a commercial cellulase mixture) up to fourfold on a range of residues from sugar cane bagasse pretreated at the laboratory (using acidified ethylene carbonate/ethylene glycol, 1-butyl-3-methylimidazolium chloride, and ball-milling) and pilot (dilute sodium hydroxide and glycerol/hydrochloric acid steam explosion) scales. We have demonstrated using tap water as a solvent (under conditions that mimic an industrial process) extraction of about 90% recombinant cellobiohydrolase from senescent, transgenic corn stover leaf that had minimal tissue disruption. Conclusions The accumulation of recombinant cellobiohydrolase in senescent, transgenic corn stover leaf is a viable strategy to reduce the saccharification cost associated with the production of fermentable sugars from pretreated biomass. We envisage an industrial-scale process in which transgenic plants provide both fibre and biomass-degrading enzymes for pretreatment and enzymatic hydrolysis, respectively.

Organosolv pretreatment of plant biomass for enhanced enzymatic saccharification

The combination of dwindling petroleum reserves and population growth make the development of renewable energy and chemical resources more pressing than ever before. Plant biomass is the most abundant renewable source for energy and chemicals. Enzymes can selectively convert the polysaccharides in plant biomass into simple sugars which can then be upgraded to liquid fuels and platform chemicals using biological and/or chemical processes. Pretreatment is essential for efficient enzymatic saccharification of plant biomass and this article provides an overview of how organic solvent (organosolv) pretreatments affect the structure and chemistry of plant biomass, and how these changes enhance enzymatic saccharification. A comparison between organosolv pretreatments utilizing broadly different classes of solvents (i.e., low boiling point, high boiling point, and biphasic) is presented, with a focus on solvent recovery and formation of by-products. The reaction mechanisms that give rise to these by-products are investigated and strategies to minimize by-product formation are suggested. Finally, process simulations of organosolv pretreatments are compared and contrasted, and discussed in the context of an industrial-scale plant biomass to fermentable sugar process.

Pretreatment of sugarcane bagasse by acid-catalysed process in aqueous ionic liquid solutions

A biomass pretreatment process was developed using acidified ionic liquid (IL) solutions containing 10-30% water. Pretreatment of sugarcane bagasse at 130°C for 30min by aqueous 1-butyl-3-methylimidazolium chloride (BMIMCl) solution containing 1.2% HCl resulted in a glucan digestibility of 94-100% after 72h of enzymatic hydrolysis. HCl was found to be a more effective catalyst than H(2)SO(4) or FeCl(3). Increasing acid concentration (from 0.4% to 1.2%) and reaction temperature (from 90 to 130°C) increased glucan digestibility. The glucan digestibility of solid residue obtained with the acidified BMIMCl solution that was re-used for three times was >97%. The addition of water to ILs for pretreatment could significantly reduce IL solvent costs and allow for increased biomass loadings, making the pretreatment by ILs a more economic proposition.

Effects of pH on pretreatment of sugarcane bagasse using aqueous imidazolium ionic liquids

Pretreatments of sugarcane bagasse for saccharification using different acid-catalysed imidazolium IL solutions (containing 20% water) at 130 °C for 30 min were investigated. At the same solution pH, pretreatment effectiveness in terms of glucan digestibility, delignification and xylan removal were similar for aqueous 1-butyl-3-methylimidazolium methane sulfonate (BMIMCH3SO3), 1-butyl-3-methylimidazolium methyl sulfate (BMIMCH3SO4), 1-ethyl-3-methylimidazolium chloride (EMIMCl) and 1-butyl-3-methylimidazolium chloride (BMIMCl). Decreasing solution pH of aqueous IL systems from 6.0 to 0.4 increased bagasse delignification and xylan removal, and as a result, improved glucan digestibility. The glucan digestibilities for bagasse samples pretreated by IL solutions with pH ≤ 0.9 were > 90% after 72 h of enzymatic hydrolysis. Without pH adjustment, the effectiveness of these aqueous IL solutions (except BMIMCH3SO3 because of its low natural pH of 0.9) to deconstruct the biomass was poor and the glucan digestibilities of pretreated bagasse samples were < 20%. These results show that pretreatment effectiveness of aqueous imidazolium ILs can simply be estimated from solution pH rather than hydrogen bond basicity (β) of the IL solution.

Low temperature pretreatment of sugarcane bagasse at atmospheric pressure using mixtures of ethylene carbonate and ethylene glycol

A low temperature lignocellulose pretreatment process was developed using acid-catalysed mixtures of alkylene carbonate and alkylene glycol. Pretreatment of sugarcane bagasse with mixtures of ethylene carbonate (EC) and ethylene glycol (EG) was more effective than that with mixtures of propylene carbonate (PC) and propylene glycol (PG). These mixtures were more effective than the individual components in making bagasse cellulose more amenable to cellulase digestion. Glucan digestibilities of ≥87% could be achieved with a wide range of EC to EG ratios from 9:1 to 1:1 (w/w). Pretreatment of bagasse by the EC/EG mixture with a ratio of 4:1 in the presence of 1.2% H2SO4 at 90 °C for 30 min led to the highest glucan enzymatic digestibility of 93%. The high glucan digestibilities obtained under these acidic conditions were due to (a) the ability of alkylene carbonate to cause significant biomass size reduction, (b) the ability of alkylene glycol to cause biomass defibrillation, (c) the ability of alkylene carbonate and alkylene glycol to remove xylan and lignin, and (d) the magnified above attributes in the mixtures of alkylene carbonate and alkylene glycol.

Pretreatment of sugarcane bagasse by acidified aqueous polyol solutions

Pretreatments of sugarcane bagasse by three high boiling-point polyol solutions were compared in acid-catalysed processes. Pretreatments by ethylene glycol (EG) and propylene glycol solutions containing 1.2 % H2SO4 and 10 % water at 130 °C for 30 min removed 89 % lignin from bagasse resulting in a glucan digestibility of 95 % with a cellulase loading of ~20 FPU/g glucan. Pretreatment by glycerol solution under the same conditions removed 57 % lignin with a glucan digestibility of 77 %. Further investigations with EG solutions showed that increases in acid content, pretreatment temperature and time, and decrease in water content improved pretreatment effectiveness. A good linear correlation of glucan digestibility with delignification was observed with R2 = 0.984. Bagasse samples pretreated with EG solutions were characterised by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction, which confirmed that improved glucan enzymatic digestibility is mainly due to delignification and defibrillation of bagasse. Pretreatment by acidified EG solutions likely led to the formation of EG-glycosides. Up to 36 % of the total lignin was recovered from pretreatment hydrolysate, which may improve the pretreatment efficiency of recycled EG solution.

Glycerol carbonate as green solvent for pretreatment of sugarcane bagasse

Background Pretreatment of lignocellulosic biomass is a prerequisite for effective saccharification to produce fermentable sugars. We have previously reported an effective low temperature (90 °C) process at atmospheric pressure for pretreatment of sugarcane bagasse with acidified mixtures of ethylene carbonate (EC) and ethylene glycol (EG). In this study, “greener” solvent systems based on acidified mixtures of glycerol carbonate (GC) and glycerol were used to treat sugarcane bagasse and the roles of each solvent in deconstructing biomass were determined. Results Pretreatment of sugarcane bagasse at 90 °C for only 30 min with acidified GC produced a solid residue having a glucan digestibility of 90% and a glucose yield of 80%, which were significantly higher than a glucan digestibility of 16% and a glucose yield of 15% obtained for bagasse pretreated with acidified EC. Biomass compositional analyses showed that GC pretreatment removed more lignin than EC pretreatment (84% vs 54%). Scanning electron microscopy (SEM) showed that fluffy and size-reduced fibres were produced from GC pretreatment whereas EC pretreatment produced compact particles of reduced size. The maximal glucan digestibility and glucose yield of GC/glycerol systems were about 7% lower than those of EC/ethylene glycol (EG) systems. Replacing up to 50 wt% of GC with glycerol did not negatively affect glucan digestibility and glucose yield. The results from pretreatment of microcrystalline cellulose (MCC) showed that (1) pretreatment with acidified alkylene glycol (AG) alone increased enzymatic digestibility compared to pretreatments with acidified alkylene carbonate (AC) alone and acidified mixtures of AC and AG, (2) pretreatment with acidified GC alone slightly increased, but with acidified EC alone significantly decreased, enzymatic digestibility compared to untreated MCC, and (3) there was a good positive linear correlation of enzymatic digestibility of treated and untreated MCC samples with congo red (CR) adsorption capacity. Conclusions Acidified GC alone was a more effective solvent for pretreatment of sugarcane bagasse than acidified EC alone. The higher glucose yield obtained with GC-pretreated bagasse is possibly due to the presence of one hydroxyl group in the GC molecular structure, resulting in more significant biomass delignification and defibrillation, though both solvent pretreatments reduced bagasse particles to a similar extent. The maximum glucan digestibility of GC/glycerol systems was less than that of EC/EG systems, which is likely attributed to glycerol being less effective than EG in biomass delignification and defibrillation. Acidified AC/AG solvent systems were more effective for pretreatment of lignin-containing biomass than MCC.

Microbial oil production from sugarcane bagasse hydrolysates by oleaginous yeast and filamentous fungi

This study investigated the potential use of sugarcane bagasse as a feedstock for oil production through microbial cultivation. Bagasse was subjected to dilute acid pretreatment with 0.4 wt% H2SO4 (in liquid) at a solid/liquid ratio of 1:6 (wt/wt) at 170 °C for 15 min, followed by enzymatic hydrolysis of solid residue. The liquid fractions of the pretreatment process and the enzymatic hydrolysis process were detoxified and used as liquid hydrolysate (SCBLH) and enzymatic hydrolysate (SCBEH) for the microbial oil production by oleaginous yeast (Rhodotorula mucilaginosa) and filamentous fungi (Aspergillus oryzae and Mucor plumbeus). The results showed that all strains were able to grow and produce oil from bagasse hydrolysates. The highest oil concentrations produced from bagasse hydrolysates were by M. plumbeus at 1.59 g/L (SCBLH) and 4.74 g/L (SCBEH). The microbial oils obtained have similar fatty acid compositions to vegetable oils, indicating that the oil can be used for the production of second generation biodiesel. On the basis of oil yields obtained by M. plumbeus, from 10 million t (wet weight) of bagasse generated annually from sugar mills in Australia, it is estimated that the total biodiesel that could be produced would be equivalent to about 9% of Queensland’s diesel consumption.

A novel population balance model for the dilute acid hydrolysis of hemicellulose

Acid hydrolysis is a popular pretreatment for removing hemicellulose from lignocelluloses in order to produce a digestible substrate for enzymatic saccharification. In this work, a novel model for the dilute acid hydrolysis of hemicellulose within sugarcane bagasse is presented and calibrated against experimental oligomer profiles. The efficacy of mathematical models as hydrolysis yield predictors and as vehicles for investigating the mechanisms of acid hydrolysis is also examined. Experimental xylose, oligomer (degree of polymerisation 2 to 6) and furfural yield profiles were obtained for bagasse under dilute acid hydrolysis conditions at temperatures ranging from 110C to 170C. Population balance kinetics, diffusion and porosity evolution were incorporated into a mathematical model of the acid hydrolysis of sugarcane bagasse. This model was able to produce a good fit to experimental xylose yield data with only three unknown kinetic parameters ka, kb and kd. However, fitting this same model to an expanded data set of oligomeric and furfural yield profiles did not successfully reproduce the experimental results. It was found that a ``hard-to-hydrolyse'' parameter, $\alpha$, was required in the model to ensure reproducibility of the experimental oligomer profiles at 110C, 125C and 140C. The parameters obtained through the fitting exercises at lower temperatures were able to be used to predict the oligomer profiles at 155C and 170C with promising results. The interpretation of kinetic parameters obtained by fitting a model to only a single set of data may be ambiguous. Although these parameters may correctly reproduce the data, they may not be indicative of the actual rate parameters, unless some care has been taken to ensure that the model describes the true mechanisms of acid hydrolysis. It is possible to challenge the robustness of the model by expanding the experimental data set and hence limiting the parameter space for the fitting parameters. The novel combination of ``hard-to-hydrolyse'' and population balance dynamics in the model presented here appears to stand up to such rigorous fitting constraints.

Non-alcoholic fatty liver disease : can ultrasound assist in early diagnosis of prediabetes and delay progression to type2 diabetes mellitus

Non Alcoholic Fatty Liver Disease (NAFLD) is a condition that is frequently seen but seldom investigated. Until recently, NAFLD was considered benign, self-limiting and unworthy of further investigation. This opinion is based on retrospective studies with relatively small numbers and scant follow-up of histology data. (1) The prevalence for adults, in the USA is, 30%, and NAFLD is recognized as a common and increasing form of liver disease in the paediatric population (1). Australian data, from New South Wales, suggests the prevalence of NAFLD in “healthy” 15 year olds as being 10%.(2) Non-alcoholic fatty liver disease is a condition where fat progressively invades the liver parenchyma. The degree of infiltration ranges from simple steatosis (fat only) to steatohepatitis (fat and inflammation) steatohepatitis plus fibrosis (fat, inflammation and fibrosis) to cirrhosis (replacement of liver texture by scarred, fibrotic and non functioning tissue).Non-alcoholic fatty liver is diagnosed by exclusion rather than inclusion. None of the currently available diagnostic techniques -liver biopsy, liver function tests (LFT) or Imaging; ultrasound, Computerised tomography (CT) or Magnetic Resonance Imaging (MRI) are specific for non-alcoholic fatty liver. An association exists between NAFLD, Non Alcoholic Steatosis Hepatitis (NASH) and irreversible liver damage, cirrhosis and hepatoma. However, a more pervasive aspect of NAFLD is the association with Metabolic Syndrome. This Syndrome is categorised by increased insulin resistance (IR) and NAFLD is thought to be the hepatic representation. Those with NAFLD have an increased risk of death (3) and it is an independent predictor of atherosclerosis and cardiovascular disease (1). Liver biopsy is considered the gold standard for diagnosis, (4), and grading and staging, of non-alcoholic fatty liver disease. Fatty-liver is diagnosed when there is macrovesicular steatosis with displacement of the nucleus to the edge of the cell and at least 5% of the hepatocytes are seen to contain fat (4).Steatosis represents fat accumulation in liver tissue without inflammation. However, it is only called non-alcoholic fatty liver disease when alcohol - >20gms-30gms per day (5), has been excluded from the diet. Both non-alcoholic and alcoholic fatty liver are identical on histology. (4).LFT’s are indicative, not diagnostic. They indicate that a condition may be present but they are unable to diagnosis what the condition is. When a patient presents with raised fasting blood glucose, low HDL (high density lipoprotein), and elevated fasting triacylglycerols they are likely to have NAFLD. (6) Of the imaging techniques MRI is the least variable and the most reproducible. With CT scanning liver fat content can be semi quantitatively estimated. With increasing hepatic steatosis, liver attenuation values decrease by 1.6 Hounsfield units for every milligram of triglyceride deposited per gram of liver tissue (7). Ultrasound permits early detection of fatty liver, often in the preclinical stages before symptoms are present and serum alterations occur. Earlier, accurate reporting of this condition will allow appropriate intervention resulting in better patient health outcomes. References 1. Chalasami N. Does fat alone cause significant liver disease: It remains unclear whether simple steatosis is truly benign. American Gastroenterological Association Perspectives, February/March 2008 www.gastro.org/wmspage.cfm?parm1=5097 Viewed 20th October, 2008 2. Booth, M. George, J.Denney-Wilson, E: The population prevalence of adverse concentrations with adiposity of liver tests among Australian adolescents. Journal of Paediatrics and Child Health.2008 November 3. Catalano, D, Trovato, GM, Martines, GF, Randazzo, M, Tonzuso, A. Bright liver, body composition and insulin resistance changes with nutritional intervention: a follow-up study .Liver Int.2008; February 1280-9 4. Choudhury, J, Sanysl, A. Clinical aspects of Fatty Liver Disease. Semin in Liver Dis. 2004:24 (4):349-62 5. Dionysus Study Group. Drinking factors as cofactors of risk for alcohol induced liver change. Gut. 1997; 41 845-50 6. Preiss, D, Sattar, N. Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogenesis and treatment considerations. Clin Sci.2008; 115 141-50 7. American Gastroenterological Association. Technical review on nonalcoholic fatty liver disease. Gastroenterology.2002; 123: 1705-25

The “Iranian ART Revolution”: infertility, assisted reproductive technology, and third-party donation in the Islamic Republic of Iran

Infertility is a social onus for women in Iran, who are expected to produce children early within marriage. With its estimated 1.5 million infertile couples, Iran is the only Muslim country in which assisted reproductive technologies (ARTs) using donor gametes and embryos have been legitimized by religious authorities and passed into law. Th is has placed Iran, a Shia-dominant country, in a unique position vis-à-vis the Sunni Islamic world, where all forms of gamete donation are strictly prohibited. In this article, we first examine the “Iranian ART revolution” that has allowed donor technologies to be admitted as a form of assisted reproduction. Then we examine the response of Iranian women to their infertility and the profound social pressures they face. We argue that the experience of infertility and its treatment are mediated by women’s socioeconomic position within Iranian society. Many women lack economic access to in vitro fertilization (IVF) technologies and fear the moral consequences of gamete donation. Thus, the benefits of the Iranian ART revolution are mixed: although many Iranian women have been able to overcome their infertility through ARTs, not all women’s lives are improved by these technologies.