High Enzyme Dosages Research Articles

Technical feasibility and modeling of enzymatic pre-treatments of organic fraction of municipal solid waste to improve anaerobic digestion

The study explored the combined effects of enzymatic pre-treatment and anaerobic digestion (AD) on the organic fraction of municipal solid wastes (OFMSW) through experimental and multicriteria decision-making approaches. Five enzymes (UPP2, MPCS, USC4, USE2, and A. niger) and their dosages were studied. AD parameters included two inoculum origins (waste active sludge - WAS - and cow-agricultural sludge - CAS), the substrate: inoculum (SI) ratio, and inoculum incubation time (INOC). Desirability functions were used to optimize the multiple experimental responses simultaneously by converting each of them into values from 0 (unacceptable) to 1 (completely acceptable) and then combining these into a global desirability (D). D highlighted that higher enzyme dosages, INOC, and SI, improved AD performances, with optimal DOSE (at the highest level adopted for each enzyme) and INOC (5–10 d). AD tests with the five enzymes increased CH4 production by 10–13%v/v compared to untreated OFMSW. For UPP2 and MPCS, increasing DOSE boosted the biogas production, while increasing INOC enhanced the CH4 content. MPCS reached the highest efficiency (478. 43 NL CH4/kg VS with CAS, SI = 2:1, INOC = 10 d), followed by UPP2. Furthermore, higher INOC reduced A. niger doses, increasing CH4 production by 9%v/v compared to literature, with 5–10 d INOC (452.86 NL s/kg VS with WAS, SI = 2:1).

Intensification of corn fiber saccharification using a tailor made enzymatic cocktail

The transition from an economic model based on resource extraction to a more sustainable and circular economy requires the development of innovative methods to unlock the potential of raw materials such as lignocellulosic biomasses. Corn fiber differs from more traditional lignocellulosic biomasses due to its high starch content, which provides additional carbohydrates for fermentation-based biomanufacturing processes. Due to its unique chemical composition, this study focused on the development of a tailor made enzymatic cocktail for corn fiber saccharification into monosaccharides. Three commercially available hydrolytic enzymes (Cellic® CTec2, Pentopan® Mono BG, and Termamyl® 300 L) were combined to hydrolyze the polysaccharide structure of the three main carbohydrate fractions of corn fiber (cellulose, hemicellulose and starch, respectively). Prior to saccharification, corn fiber was submitted to a mild hydrothermal pretreatment (30 min at 100 °C). Then, two experimental designs were used to render an enzymatic cocktail capable of providing efficient release of monosaccharides. Using 60 FPU/g DM of Cellic® CTec2 and 4.62 U/g DM of Termamyl® 300 L, without addition of Pentopan® Mono BG, resulted in the highest efficiencies for glucose and xylose release (66% and 30%, respectively). While higher enzyme dosages could enhance the saccharification efficiency, adding more enzymes would have a more pronounced effect on the overall process costs rather than in increasing the efficiency for monosaccharides release. The results revealed that the recalcitrance of corn fiber poses a problem for its full enzymatic degradation. This fact combined with the unique chemical composition of this material, justify the need for developing a tailor made enzymatic cocktail for its degradation. However, attention should also be given to the pretreatment step to reduce even more the recalcitrance of corn fiber and improve the performance of the tailored cocktail, as a consequence.

Recent advances in substrate-enzyme interactions facilitating efficient biodegradation of lignocellulosic biomass: A review

Lignocellulosic biorefinery has long been recognized as a potential strategy for sustainable production of biofuels, materials and chemicals. These productions in many cases rely on pretreatment and enzymatic hydrolysis of lignocellulosic carbohydrates, whereas the low degradation efficiency and high enzyme dosage are challenging bottlenecks of economically industrial production. The complex recalcitrant nature of lignocellulose requires a well-matched enzyme cocktail to achieve complete and efficient degradation. Numerous studies have investigated the relationship between biomass components, structural properties and enzymes synergistic action in the past decades. This article aims to present a comprehensive review on this subject by introducing recent advances in substrate modification techniques, underlying mechanisms of enzyme synergism and governing factors in structure-activity interaction. In addition, newly developed modellings and multi-omic analysis techniques for preparing commercial enzyme preparations are introduced, revealing possible avenues for achieving economic viability of lignocellulose bioconversion.

Effective Fermentation of Sugarcane Bagasse Whole Slurries Using Robust Xylose-Capable Saccharomyces cerevisiae

The pre-treatment of lignocellulose material towards cellulosic bioethanol production releases microbial inhibitors that severely limit the fermentation ability of Saccharomyces cerevisiae. This study evaluated to what degree robust xylose-capable strains may improve the fermentability of non-detoxified sugarcane bagasse (SCB) slurries derived from steam explosion (StEX) and further compared this to slurries derived from ammonia fibre expansion (AFEX) pre-treatment. Initial screening in separate hydrolyses and co-fermentation processes using StEx-SCB hydrolysates identified S. cerevisiae TP-1 and CelluXTM4 with higher xylose consumption (≥ 88%) and ethanol concentrations (≥ 50 g/L), and ethanol metabolic yields (≥89% relative to theoretical maximum), even in the presence of approximately 8 g/L of acetic acid. Under industrially relevant pre-hydrolysis simultaneous saccharification and co-fermentation (PSSCF) conditions of high solids loading (15%, w/w) and low enzyme dosage (8 mg protein per gram untreated biomass), the fermentation of StEx-treated SCB whole slurry achieved ethanol yields of 208 and 224 L per Mg raw dry SCB using S. cerevisiae TP-1 and CelluXTM4, respectively. Under the same solids loading and enzyme dosages, the PSSCF of ammonia fibre expansion (AFEXTM) pre-treated SCB achieved ethanol yields of 234 and 251 L per Mg raw dry SCB using TP-1 and CelluXTM4, respectively. The study achieved non-detoxified whole-slurry co-fermentation using StEx pre-treated SCB, with higher ethanol yields than previously reported, by utilising robust xylose-capable strains. The results of this work provide insights into the potential use of inhibitor-tolerant S. cerevisiae strains TP-1 and CelluXTM4 as ethanologens for the fermentation of steam-exploded and undetoxified SCB whole slurries.

Influence of mixing speed, solids concentration and enzyme dosage on dry solids yield and protein recovery during enzymatic hydrolysis of sardine (Sardina pilchardus) processing by-products using Alcalase 2.4L: a multivariable optimisation approach

Yield and protein recovery are important variables for process design, which cannot be achieved using degree of hydrolysis (DH). Even though it is known that mixing speed and solids concentration affect mass and energy transfer in bioprocess reaction, previous research has not provided a clear relationship between these two variables and their effect on yield of dry solids and protein recovery during enzyme hydrolysis. The yield of dry solids and protein recovery from enzyme hydrolysis of sardine processing by-products was compared at different levels of mixing speed (100–300 rpm), solids concentration (26–50%) and enzyme dosage (1.318–4.682%). Results showed that low mixing speed (100 rpm), low solids concentration (26–30.8%) and high enzyme dosage (4.682%) optimised degree of hydrolysis to 25.7%, yield of dry solids to 69.1% and protein recovery to 83.0%. Under these conditions, protein loss to emulsion and sludge were minimised to 5.82% and 11.2%, respectively. Although low solids concentration resulted in high solids yield and high protein recovery due to favourable mass transfer effects, hydrolysing material under these conditions will come at a significant cost of larger equipment designs and energy cost for mixing and downstream processing because of the large volume of water to be handled. There is therefore a need for a holistic approach to enzyme hydrolysis optimisation studies, with downstream processing in mind. The significant findings of this study show the impact of process variables and their interaction, particularly solids concentration, on process performance using multivariable optimisation.

High Solid and Low Cellulase Enzymatic Hydrolysis of Cardoon Stems Pretreated by Acidified γ-Valerolactone/Water Solution

Lignocellulosic biomass is a nonedible matrix that can be efficiently exploited as feedstock in an integrated biorefinery after a proper pretreatment. An organosolv pretreatment using an acidified γ-valerolactone (GVL)/water solution was proposed to improve the cellulose enrichment and enzymatic saccharification of cardoon (Cynara cardunculus L.) stems. At the optimal pretreatment condition (140 °C, 0.6 GVL/water, and 2.24% H2SO4), xylan was efficiently removed from the cardoon, and up to 50% of its content was recovered in the aqueous fraction, while 86% of the cellulose was retained in the solid fraction. The resulting cardoon pulp showed a cellulose content of 91.5% and an enzymatic digestibility of 100%. An overall glucose production of 37.17 g/100 g raw material (90% theoretical maximum) was obtained using high solid loading (20% w/w) and a high enzyme dosage (60 FPU/g cellulose). At a low enzyme dosage, glucose concentrations of 169 g/L and 210 g/L were achieved using 10 FPU/g cellulose and 20 FPU/g cellulose, respectively. Therefore, an organosolv pretreatment can be an effective process for producing cellulose-enriched pulp with enhanced enzymatic digestibility from cardoon stems, providing a promising option for green lignocellulosic biorefineries that aim to produce high concentrations of glucose with low cellulase addition.

Cellulolytic enzymes behavior in delignified green coconut residues and enzymatic hydrolysis with enzyme recovery

Lignocellulosic bioconversion to obtain fermentable sugars often requires high enzyme dosages, increasing the total production cost. This study investigated the adsorption, desorption, and recycling of cellulases in enzymatic hydrolysis experiments using pretreated green coconut shell as substrate. The green coconut shell (GCS) was subjected to different pretreatments including dilute acid, alkaline, and two-stage acid-alkaline pretreatments. Adsorption and desorption studies involving cellulases from Trichoderma reesei ATCC 26921 and Cellic Ctec2 (10 FPU/g, initial dosage) with 5% solid loading showed that alkaline and two-stage acid-alkaline pretreatments were more suitable for enzyme recycling, because they showed lower values of cellulase adsorption capacity (48 % and 69 %) and higher values of cellulase recovery by desorption without Tween 80 addition (reaching 50 % using two-stage acid-alkaline pretreated GCS). Cellulases bound to the solid residue and dissolved in the supernatant after enzymatic hydrolysis can be reused for a new enzymatic hydrolysis process without compromising the sugar yield results. Using GCS as a substrate, the addition of solid residue and the supernatant from the previous step of adsorption of cellulases achieved a satisfactory sugar yield g/g (73.0 % for LCM1 and 60.0 % for LCM3) using less enzyme (just using recycled enzyme with no new enzyme added to the system) than the control case. Thus, it was possible to recycle the cellulases in two successive hydrolysis cycles with reduced enzyme load to achieve the same reducing sugar yields (up to 84 %), enabling cheaper and more efficient hydrolysis of lignocellulosic biomass.

High solid loading enzymatic hydrolysis of acetic acid-peroxide/acetic acid pretreated poplar and cellulase recycling

High solid loading saccharification is the premise of preparing high-concentration sugar which is beneficial to bioethanol production, but the limited sugar concentration and high enzyme dosage are two challenges. In this work, the glucan-rich acetic acid-hydrogen peroxide/acetic acid (AC-HPAC)-pretreated poplar (85.8%) were prepared for enzymatic hydrolysis at 10%-40% solid loading and the strategies for reducing cellulase dosage were explored. Results showed that the maximum glucose concentration reached to 250.8 g/L at 40% solid loading, which was the highest concentration in previous literatures. As the solid loading was 20%, the addition of Tween 80 saved 50% of cellulase and the recycling of unhydrolyzed residue (0.2 g/g DM) saved another 25% of cellulase, resulting in 152.2 g/L of glucose concentration with yield of 79.9%. This work showed potential of poplar to produce the high concentration glucose solution with low enzyme loading through the recycling of enzyme bound onto unhydrolyzed residue.

Modulation of Nitric Oxide Bioavailability Attenuates Ischemia-Reperfusion Injury in Type II Diabetes

Diabetes mellitus increases the susceptibility of free tissue transplantations to ischemia-reperfusion injury. The aim of this study was to enhance nitric oxide (NO) bioavailability through exogenous NO synthase and the substrate L-arginine to attenuate ischemia reperfusion-induced alterations in a type 2 diabetes rodent model. Sixty-four Wistar rats were divided into 8 experimental groups. Type 2 diabetes was established over 3 months with a combination of a high-fat diet and streptozotocin. A vascular pedicle isolated rat skin flap model that underwent 3 h of ischemia was used. At 30 min before ischemia, normal saline, endothelial NOSs (eNOSs), inducible NOSs, neuronal NOSs (1 and 2 IU), and L-arginine (50 mg/kg body weight) were administered by intravenous infusion alone or in combination. Ischemia-reperfusion-induced alterations were measured 5 days after the operation. The three isoforms of NOS significantly increased the flap vitality rate (VR) between 20% and 28% as compared to the control group (3%). Sole L-arginine administration increased the VR to 33%. The combination of L-arginine with NOS resulted in a further increase in flap VRs (39%-50%). Best results were achieved with the combination of eNOS and L-arginine (50%). An increase in enzyme dosage led to decreased VRs in all NOS isoforms alone and even in combination with L-arginine. Modulation of NO bioavailability through the exogenous application of NOSs and L-arginine significantly attenuated ischemia-reperfusion-induced alterations in a type 2 diabetic skin flap rat model. The combination of enzyme and substrate result in the highest VRs. Higher enzyme dosage seems to be less effective. This pharmacological preconditioning could be an easy and effective interventional strategy to support the conversion of L-arginine to NO in ischemic and in type 2 diabetic conditions.

Drying Effect on Enzymatic Hydrolysis of Cellulose Associated with Porosity and Crystallinity

The effect of drying on the enzymatic hydrolysis of cellulose was determined by analysis of porosity and crystallinity. Fiber hornification induced by drying produced an irreversible reduction in pore volume due to shrinkage and pore collapse, and the decrease in porosity inhibited enzymatic hydrolysis. The drying effect index (DEI) was defined as the difference in enzymatic digestibility between oven- and never-dried pulp, and it was determined that more enzymes caused a higher DEI at the initial stage of enzymatic hydrolysis and the highest DEI was also observed at the earlier stages with higher enzyme dosage. However, there was no significant difference in the DEI with less enzymes because cellulose conversion to sugars during hydrolysis did not enhance enzymatic hydrolysis due to the decrease in enzyme activity. The water retention value (WRV) and Simons’ staining were used to measure pore volume and to investigate the cause of the decrease in enzymatic hydrolysis. A decrease in enzyme accessibility induced by the collapse of enzymes’ accessible larger pores was determined and this decreased the enzymatic hydrolysis. However, drying once did not cause any irreversible change in the crystalline structure, thus it seems there is no correlation between enzymatic digestibility and crystalline structure.

Techno-economic analysis of a liquid hot water pretreated switchgrass biorefinery: Effect of solids loading and enzyme dosage on enzymatic hydrolysis

Material and energy results were used in a techno-economic model to analyze the effect of different parameters and configurations on the economics of the process of a liquid hot water pretreated switchgrass (Panicum virgatum L.) biorefinery. Previous experimental results for switchgrass composition and its cellulose enzymatic hydrolysis at different high solids content and enzyme dosages were used in order to obtain a more realistic analysis. The minimum ethanol selling price (MESP) obtained for a facility that produces only ethanol and electricity was within the expected price range for advanced alcohol fuels and could compete with oil prices above $100 per barrel. The MESP was lower for a biorefinery scenario that produces furfural, acetic acid, and formic acid as high-value co-products. The MESP was sensitive to plant size and to switchgrass composition. Enzyme dosage, solids content, and hydrolysis and fermentation efficiencies were the operating parameters with a higher impact on the MESP. Experimental results were combined with the techno-economic models to find correlations between the MESP, and solids content and enzyme dosage. The conditions necessary for a high efficiency of hydrolysis and glucose concentration are not necessarily those that produce an economic optimum (lower cost). The enzyme dosage that minimizes MESP is not always associated with the optimal hydrolysis efficiency (e.g., 95%). The best conditions identified through optimization were an enzyme dosage of 37 mgprotein gglucan−1 and a solids content of 21%, leading to the lowest MESP of $0.84 L-1 (for an enzyme cost of $4.5 kgprotein−1).

Ethanol production potential from AFEX\u2122 and steam-exploded sugarcane residues for sugarcane biorefineries

BackgroundExpanding biofuel markets are challenged by the need to meet future biofuel demands and mitigate greenhouse gas emissions, while using domestically available feedstock sustainably. In the context of the sugar industry, exploiting under-utilized cane leaf matter (CLM) in addition to surplus sugarcane bagasse as supplementary feedstock for second-generation ethanol production has the potential to improve bioenergy yields per unit land. In this study, the ethanol yields and processing bottlenecks of ammonia fibre expansion (AFEX™) and steam explosion (StEx) as adopted technologies for pretreating sugarcane bagasse and CLM were experimentally measured and compared for the first time.ResultsEthanol yields between 249 and 256 kg Mg−1 raw dry biomass (RDM) were obtained with AFEX™-pretreated sugarcane bagasse and CLM after high solids loading enzymatic hydrolysis and fermentation. In contrast, StEx-pretreated sugarcane bagasse and CLM resulted in substantially lower ethanol yields that ranged between 162 and 203 kg Mg−1 RDM. The ethanol yields from StEx-treated sugarcane residues were limited by the aggregated effect of sugar degradation during pretreatment, enzyme inhibition during enzymatic hydrolysis and microbial inhibition of S. cerevisiae 424A (LNH-ST) during fermentation. However, relatively high enzyme dosages (> 20 mg g−1 glucan) were required irrespective of pretreatment method to reach 75% carbohydrate conversion, even when optimal combinations of Cellic® CTec3, Cellic® HTec3 and Pectinex Ultra-SP were used. Ethanol yields per hectare sugarcane cultivation area were estimated at 4496 and 3416 L ha−1 for biorefineries using AFEX™- or StEx-treated sugarcane residues, respectively.ConclusionsAFEX™ proved to be a more effective pretreatment method for sugarcane residues relative to StEx due to the higher fermentable sugar recovery and enzymatic hydrolysate fermentability after high solids loading enzymatic hydrolysis and fermentation by S. cerevisiae 424A (LNH-ST). The identification of auxiliary enzyme activities, adequate process integration and the use of robust xylose-fermenting ethanologens were identified as opportunities to further improve ethanol yields from AFEX™- and StEx-treated sugarcane residues.

GH11 xylanase increases prebiotic oligosaccharides from wheat bran favouring butyrate-producing bacteria in vitro

Alternative solutions to optimise intestinal health in monogastric animals have become essential since the ban of antimicrobials in animal feed. In this study, the prebiotic potential of a commercial feed GH11 xylanase was investigated in vitro. Enzymatic degradation of arabinoxylan (AX), the substrate present in wheat bran cell walls, was visualised using immuno-microscopy techniques. The arabinoxylooligosaccharides (AXOS) generated by the enzyme were analysed by non-starch polysaccharide (NSP) analysis, mass spectrometry (MS) and carbohydrate chromatography to investigate how AXOS glycan complexity and enzyme dosage may affect fermentation patterns in a wheat-based diet. Using a 10mg EP/kg dosage of xylanase, AXOS with an average degree of polymerisation (avDP) of 10 were generated, while using a higher enzyme dosage (50mg EP/kg) avDP shifted to 4–8. For both enzyme concentrations, AXOS had an arabinose/xylose ratio of ∼0.4. Wheat bran incubated without or with xylanase was simultaneously fermented by broiler cecal bacteria in vitro and short chain fatty acid production was monitored. A small (but significant) increase in butyrate production by addition of xylanase was shown to be dose-dependent and increased by 2mM (P<0.05) compared to control by adding 50mg EP/kg enzyme dosage. Butyrate-producing bacterial genera Faecalibacterium and Intestinimonas were significantly increased in fermentation reactions of wheat bran with GH11 xylanase addition while Bacteroidetes levels were significantly lowered. Supernatants from fermentation reactions of wheat bran incubated with and without xylanase and cecal microbiota were tested in an intestinal epithelial layer permeability assay using Caco-2 cells stimulated with LPS. The xylanase addition to the bran incubated with cecal content of broilers reversed LPS-induced epithelial layer resistance losses. The GH11 xylanase was able to solubilise and degrade wheat bran AX to yield low avDP AXOS that can be fermented by cecal microbiota, resulting in microbiota shifts and beneficial effects on transepithelial resistance in vitro.

The influence of the explosive decompression in steam-explosion pretreatment on the enzymatic digestibility of different biomasses.

For the production of second generation biofuels from lignocellulosic biomass, pretreatment of the biomass feedstock is necessary to overcome its recalcitrance in order to gain fermentable sugars. Due to many reasons, steam-explosion pretreatment is currently the most commonly used pretreatment method for lignocellulosic biomass on a commercial scale [S. Brethauer and M. H. Studer, CHIMIA, 2015, 69, 572-581]. In contrast to others, we showed that the explosive decompression at the end of this pretreatment step can have a positive influence on the enzymatic digestibility of softwood, especially in combination with high enzyme dosages [T. Pielhop, et al., Biotechnology for Biofuels, 2016, 9, 152]. In this study, the influence of the explosive decompression on the enzymatic digestibility of hardwood and herbaceous plants was systematically studied. Beech and corn stover were pretreated under different pretreatment conditions and enzymatically hydrolysed with different enzyme dosages. The maximum enhancement of the digestibility of corn stover was 16.53% after a 2.5 min pretreatment step at 15 barg steam pressure. For beech, a maximum relative enhancement of 58.29% after a 10 min pretreatment step at 15 barg steam pressure could be reached. With this, we show that the explosive decompression can also enhance the enzymatic cellulose digestibility of hardwood and herbaceous plants.

Production of ethanol from steam exploded triticale straw in a simultaneous saccharification and fermentation process

Abstract The cost-effective production of bioethanol requires an inexpensive feedstock that is easily hydrolysed by minimal amounts of cellulase enzymes and efficiently converted to ethanol. Steam-exploded triticale straw was investigated for ethanol production using selected commercial cellulases, with Spezyme® CP outperforming the other enzymes in terms of glucose release. Varying enzyme dosages (5, 10 and 15 FPU Spezyme® CP/g cellulose) and different solids loadings (5, 10 and 15% w/v) were evaluated in simultaneous saccarification and fermentation (SSF) experiments with Saccharomyces cerevisiae Ethanol Red® as fermenting yeast. A 15% (w/v) solids loading with 15 FPU Spezyme® CP/g cellulose produced 29.6 g/l ethanol (81.3% ethanol yield). When the high-gravity SSF was repeated in a 1-l bioreactor using 15% solids and 15 FPU Spezyme® CP/g cellulose, an ethanol concentration of 29.31 g/l (84.7% ethanol yield) was achieved after 144 h without additional β-glucosidase. Similar results have been observed by other researchers, but with higher enzyme dosages or the addition of β‐glucosidase or xylanase. This study demonstrates that triticale straw is a potential feedstock for the production of lignocellulosic bio-ethanol, and further optimisation of the SSF conditions could result in a commercially viable process for the production of bio-ethanol.

Heterologous Expression of a New Acetyl Xylan Esterase from Aspergillus niger BE-2 and its Synergistic Action with Xylan-Degrading Enzymes in the Hydrolysis of Bamboo Biomass

Efficient utilization of plant biomass by enzymatic hydrolysis is currently studied worldwide but still faces enormous challenges because of the inability to break down lignocellulosic materials with high sugar yields and low enzyme dosage. Therefore, the synergistic action between various enzymes plays an important role in reaching this goal. The synergistic cooperation between a novel acetyl xylan esterase (heterologous expressed at high levels in this study) and four other xylan-degrading enzymes (reported previously) were performed in this study. The acetyl xylan esterase (AnAxe) gene was cloned from Aspergillus niger BE-2 and expressed in Pichia pastoris GS115. The deduced amino acid (aa) sequence consisted of 304-aa and included a 23-aa signal peptide and 281-aa mature protein. The AnAxe was extracellularly expressed with a molecular weight of ca. 31 kDa. The purified AnAxe exhibited maximal specific activity of 480.2 IU/mg at pH 7.0 and 40 °C and was still thermostable below 50 °C. The metal ions used in this study and EDTA showed a slight effect on the AnAxe. A significant synergistic effect was determined between AnAxe and the other four xylan-degrading enzymes, including endo-Beta-1,4-xylanases, Beta-xylosidases, alpha-L-arabinofurano-sidases, and alpha-glucuronidases, on the degradation of bamboo biomass. The highest degree of synergism was obtained between AnAxe and endo-Beta-1,4-xylanases/Beta-xylosidases.

Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentation

Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2g/L at 66.9% and 45.5g/L at 78.2%, respectively.

Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale

Recycling of enzymes in production of lignocellulosic bioethanol has been tried for more than 30years. So far, the successes have been few and the experiments have been carried out at conditions far from those in an industrially feasible process. Here we have tested continuous enzyme recycling at demonstration scale using industrial process conditions (high dry matter content and low enzyme dosage) for a period of eight days. The experiment was performed at the Inbicon demonstration plant (Kalundborg, Denmark) capable of converting four tonnes of wheat straw per hour. 20% of the fermentation broth was recycled to the hydrolysis reactor while enzyme dosage was reduced by 5%. The results demonstrate that recycling enzymes by this method can reduce overall enzyme consumption and may also increase the ethanol concentrations in the fermentation broth. Our results further show that recycling fermentation broth also opens up the possibility of lowering the dry matter content in hydrolysis and fermentation while still maintaining high ethanol concentrations.

Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse

Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family 11 (GH11), from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6h (3.62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture.

Relationship between physicochemical properties and enzymatic hydrolysis of sugarcane bagasse varieties for bioethanol production

The structural and physicochemical characteristics are associated with resistance of plant cell walls to saccharification by enzymes. The effect of physicochemical properties on glucose yield of bagasse from different varieties of sugarcane at low and high enzyme dosages was investigated. The result showed that glucose yield at low enzyme dosage was positively linear correlated with the yield at high enzyme dosage, for both the untreated and pretreated materials. The pretreatment significantly increased the accessibility of substrates by enzyme due to the increase of internal and external surface area. Glucose yield also showed a linear correlation with dye adsorption. However, the increase in glucose yield as a result of pretreatment did not correlate with the increases in crystallinity index and decreases in degree of polymerization. The Principal Component Analysis of infrared data indicated that lignin was the main component that differentiated the varieties before and after pretreatment. These results suggested that the key differences in pretreatment responses among varieties could be mainly attributed to their differences in the internal and external surface area after pretreatment.