Effects of lignocellulosic biomass type on the economics of hydrothermal treatment of digested sludge for solid fuel and soil amendment applications.

Digested sludge is a waste stream from anaerobic digestion (AD) in wastewater treatment plants. Hydrothermal treatment (HTT) of sludge mixed with lignocellulosic biomass is an attractive approach to improve sludge dewaterability and generate value-added products. However, process economics has not been well understood. In this study, firstly, the effect of biomass type on the energy properties of hydrochars was studied. Secondly, two scenarios were simulated to evaluate the effects of biomass type on the economics (processing 50,000 tonnes of sludge per year) of HTT of digested sludge for solid fuel and soil amendment applications. The two HTT scenarios included sludge alone and sludge-biomass mixtures (four cases for four biomass feedstocks) at 180 °C for 60 min. In both scenarios, HTT liquids were returned to existing AD facilities for biomethane production to offset the energy cost of the HTT process. The results showed that the higher heating value significantly increased from 16.0-17.0 MJ kg-1 in the sludge alone case to 18.0-23.0 MJ kg-1 in sludge-biomass mixtures (except for rice husk). With the use of saved transport cost as a revenue source, HTT of sludge-biomass led to a net present value (NPV) range of AU$ 9.9-20.3 million (20 years) and an internal rate of return (IRR) range of 25.0 %-45.2 % for solid fuel application of resulting hydrochar compared to an NPV of AU$ 18.4 million and an IRR of 55.0 % from HTT of sludge alone scenario. HTT of sludge-biomass led to a NPV range of AU$ 4.5-14.5 million and an IRR range of 17.2 %-35.7 % for soil amendment application while the hydrochar from HTT of sludge alone was not recommended for soil application due to the high contents of heavy metals. This study provides useful and critical information for process scale-up and commercialization for integration into wastewater treatment plants.

Triple action of FeCl3-assisted hydrothermal treatment of digested sludge for deep dewatering.

In this study, a FeCl3-assisted hydrothermal treatment (HTT) process under mild conditions (90 °C-130 °C) was developed for deep dewatering of anaerobically digested sludge. HTT of sludge at 90 °C-130 °C with 4%-6% Fe3+ ions loading based on total sludge solids followed by mechanical dewatering reduced sludge water content from 82% to 38%-53% and sludge weight by 62%-72%. The treatment increased the flowability of sludge through reduction of apparent viscosity and disintegration of colloidal forces between sludge particles. This study unveiled that FeCl3-assisted HTT process had three mechanisms for improving sludge dewaterability and flowability. The treatment hydrolysed sludge flocs in the presence of Lewis acid FeCl3 and high temperature (90-130 °C). Fe3+ ions also improved dewaterability through the formation of double electric layers and neutralisation of surface negative charges, leading to flocculation of sludge flocs. More importantly, the hydrolysed sludge components produced during HTT process acted as reducing agents and led to in-situ generation of iron oxyhydroxide nanoparticles through reduction-oxidation reactions, further enhancing flocculation/co-precipitation of sludge flocs. The treatment reduced EPS content and changed conformational structures of EPS proteins by breaking down hydrogen bond-maintaining α-helix which led to a loose EPS protein structure and enhanced hydrophobicity and flocculability. Furthermore, the FeCl3-assisted treatment promoted immobilisation of the majority of heavy metals in the sludge matrix through co-precipitation/complexation reactions with iron species and organic/inorganic matters. This indicates that the FeCl3-assisted treatment reduced direct toxicity/bioavailability of the majority of heavy metals and the treated sludge may be suitable for land application. Overall, this study provides new insights into mechanism of FeCl3-assisted HTT process for dewaterability of anaerobically digested sludge and immobilisation of heavy metals.

Effects of hydrochar derived from hydrothermal treatment of sludge and lignocellulose mixtures on soil properties, nitrogen transformation, and greenhouse gases emissions.

In this study, hydrochar samples derived from hydrothermal treatment (HTT) of sludge and sludge-biomass mixtures were applied to a sandy soil and their effects on soil properties, soil nutrients, greenhouse gas (GHG) emissions, and soluble heavy metals were investigated. The application of untreated sludge and hydrochar derived from HTT of sludge at 180 °C led to the highest soluble nitrate, CO2 and N2O emissions, followed by the application of hydrochar samples derived from HTT of sludge-biomass mixtures at 180 °C. Although the application of hydrochar samples derived from HTT of sludge alone and sludge-biomass mixtures at 240 °C in sandy soil led to the lowest emissions of CO2 and N2O, it resulted in lower levels of soil electrical conductivity (EC), cation exchange capacity (CEC) and soluble phosphorus. The application of hydrochar samples derived from HTT at 240 °C led to the production of CH4 and lower nitrate-N contents than hydrochar samples derived from HTT at 180 °C. These results indicated that the soils containing hydrochar samples from HTT at 240 °C were anaerobic, which might inhibit the growth of plants. The application of hydrochar samples derived from HTT of sludge-biomass at 180 °C led to significantly improved contents of soil soluble phosphorus (2.56 and 2.84 g kg-1 soil) and soil nitrate-N (160.2 and 263.2 mg kg-1 soil) at the end of 60 days of incubation. However, these contents were lower than the contents of soluble phosphorus (3.71 and 4.45 g kg-1 soil) and nitrate-N (528.3 and 583.2 mg kg-1 soil) with the application of untreated sludge and sludge derived from HTT of sludge alone at 180 °C. Although more studies are needed to understand the mechanisms and effects on different soils, this study provides useful insights into the application of hydrochar derived from sludge-biomass mixture in soil.

Effects of lignocellulosic biomass type on nutrient recovery and heavy metal removal from digested sludge by hydrothermal treatment.

Sludge is a nutrient-rich organic waste generated from wastewater treatment plants. However, the application of sludge as a nutrient source is limited by its high contents of water and pollutants. In this study, the effects of biomass type on nutrient recovery and heavy metal removal from digested sludge by hydrothermal treatment (HTT) were investigated. Blending biomass with digested sludge for HTT at 180-240 °C increased the recovery of nitrogen in the treated solids. At the HTT temperature of 240 °C, HTT with hardwood sawdust led to the highest nitrogen recovery of 70.6%, compared to the lowest nitrogen recovery of 36.5% without biomass. Blending biomass slightly decreased the recovery of phosphorus compared to those without biomass. Nevertheless, the lowest phosphorus recovery of 91.3% with the use of hardwood sawdust at the HTT temperature of 240 °C was only ∼7.0% less than that without biomass. Blending biomass reduced the contents of macro-metals such as Ca, Fe, Mg and Al in treated solids but the metal contents varied with different biomasses. Regarding the heavy metals, the use of rice husk did not decrease the contents of Ni and Co while blending bagasse did not decrease the content of Cr at HTT temperatures of 210 °C and 240 °C compared to the use of other biomasses. The different effects of biomass type on nutrient recovery and heavy metals were likely related to the types and abundances of organic acids such as acetic acid, oxygen-containing functional groups such as C-OH and COOH, oxide minerals such as silica from biomasses and the overall effects of these factors. This study provides very useful information in selection of lignocellulosic biomass for HTT of sludge for nutrient recovery and heavy metal removal.

Effects of pretreatment methods on biomethane production kinetics and microbial community by solid state anaerobic digestion of sugarcane trash.

Solid state anaerobic digestion (SS-AD) of lignocellulose is effective in improving biomethane productivity but is limited by low biomass digestibility and lack of substrate-specific working microorganisms. In this study, the effects of different pretreatment methods on biomethane production by SS-AD of sugarcane trash were studied. The biomethane production, fitted to a modified Gompertz's model, predicted a maximum methane yield of 214.2 L/kg volatile solids (VS) and productivity of 6.9 L/kg VS/day from KOH-pretreated trash, respectively. Microbial community analysis showed that bacterial community was significantly associated with volatile acids and pretreatment types while archaeal community was significantly associated with methane yield. Microbial community dynamics was revealed in SS-AD. Main genera related to pretreatment method were identified and discussed. This study generated important information on SS-AD of lignocellulosic biomass pretreated by different methods, which is useful for developing bioaugmentation strategies to improve biomethane production by SS-AD.

Effect of hydrothermal treatment on deep dewatering of digested sludge: Further understanding the role of lignocellulosic biomass.

In this study, lignocellulose-assisted hydrothermal treatment (HTT) of digestated sludge was studied to further understand the role of biomass in HTT and its effect on subsequent sludge dewatering. HTT of sludge-biomass mixtures at 180 °C for 60 min at a sludge/biomass total solids (TS) ratio of 1:1 led to solid residue moistures of 36%-40% after dewatering using a hydraulic press at 24 MPa, compared to 69.5% without biomass. Further investigation showed that organic acids, especially acetic acid generated from lignocellulosic biomass hydrolysed extracellular polymeric substances (EPS), especially EPS-protein, and improved sludge dewaterability. The role of organic acids was further verified with the addition of 10.0 g/L acetic acid for HTT of sludge at 180 °C in the absence of biomass. It was also observed that in HTT of sludge with 10.0 g/L acetic acid, protein nitrogen was converted to more stable forms of nitrogen such as pyrrole­nitrogen and quaternary­nitrogen. However, HTT with acetic acid alone resulted in dewatered solids with high ash contents, which may limit their applications as soil amendments. Combination of biomass and acetic acid with a sludge/biomass TS ratio of 3:1 and acetic acid loading of 10.0 g/L at a HTT temperature of 180 °C for 60 min led to solid moistures of 50.5% with hardwood sawdust and 57.7% with sugarcane bagasse after dewatering at 3 MPa, corresponding to total weight reductions of 66.3% and 55.7%, respectively. In contrast, HTT of sludge at 180 °C for 60 min without acetic acid and biomass resulted in a solid moisture of 76.6% after dewatering at 3 MPa and a corresponding weight reduction of 49.5%. With the use of biomass and acetic acid in HTT, the treated and dewatered solids also had increased carbon content and reduced ash content. These dewatered solids may be used as potential soil amendments though the properties related to soil applications need to be considered in future studies.

Effect of ferrous iron loading on dewaterability, heavy metal removal and bacterial community of digested sludge by Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans ILS-2 was adapted in digested sludge and used to treat sludge for dewaterability improvement. Results showed that increasing ferrous iron loading increased sludge dewaterability, but the inoculation of the bioleaching strain had little effect on sludge dewaterability compared to controls without the strain. The total extracellular polymeric substances (EPS) contents of sludges with and without bioleaching treatment were similar except for bioleaching treatment at 10% ferrous iron loading (on sludge total solids) where total EPS was higher with bioleaching treatment. However, bioleaching treatment for 48 h had a notable effect on removal of heavy metals, such as Mn, Ni and Zn, especially at the high loadings of ferrous iron. In the presence of A. ferrooxidans, the removal of Ni, Mn and Zn reached 93%, 88% and 80%, respectively, at a ferrous iron loading of 21%. The sequencing of 16S rRNA genes indicated that increasing ferrous iron loadings to 15% and 21% increased the relative abundance of Acidithiobacillus, Acidocella (with A. ferrooxidans) and Carboxylicivirga (without A. ferrooxidans) but decreased the abundance of Pseudomonas and Acinetobacter after 48 h treatment. This study enhanced the understanding of the correlations between bioleaching treatment of digested sludge, sludge dewaterability, heavy metal removal and bacterial communities.

A systematic evaluation of biomethane production from sugarcane trash pretreated by different methods.

Biomethane production was systematically evaluated with sugarcane trash pretreated by liquid hot water (LHW), dilute acid (DA) and KOH solutions. Multiple linear regression analysis identified glucan in pretreated solid residue as well as C5 sugars and acetic acid in pretreatment hydrolysate as the key parameters affecting biomethane potentials. Moreover, biomethane production was best simulated using Chen & Hashimoto model with a predicted highest methane yield of 187 mL/g initial total solids (TS) based on LHW (130 °C for 15 min) and KOH (10% on trash, 150 °C for 60 min) pretreatments. KOH pretreatment led to a biomethane yield of 167 mL/g initial TS at day 25, 82%, 34% and 33% higher than those achieved with untreated and pretreated trash samples with optimal LHW and DA conditions, respectively. This study led to the identification of best kinetic model and pretreatment condition for biomethane production from sugarcane trash through a systematic evaluation.

Highly efficient production of transfructosylating enzymes using low-cost sugarcane molasses by A. pullulans FRR 5284.

Fructooligosaccharides (FOS) are a type of important prebiotics and produced by transfructosylating enzymes. In this study, sugarcane molasses was used as the substrate for production of transfructosylating enzymes by Aureobasidium pullulans FRR 5284. NaNO3 was a superior nitrogen source to yeast extract for production of transfructosylating enzymes by A. pullulans FRR 5284 and decreasing the ratio of NaNO3 to yeast extract nitrogen from 1:0 to 1:1 resulted in the reduction of the total transfructosylating activity from 109.8 U/mL to 82.5 U/mL. The addition of only 4.4 g/L NaNO3 into molasses-based medium containing 100 g/L mono- and di-saccharides resulted in total transfructosylating activity of 123.8 U/mL. Scale-up of the A. pullulans FRR 5284 transfructosylating enzyme production process from shake flasks to 1 L bioreactors improved the enzyme activity and productivity to 171.7 U/mL and 3.58 U/mL/h, 39% and 108% higher than those achieved from shake flasks, respectively. Sucrose (500 g/L) was used as a substrate for extracellular, intracellular, and total A. pullulans FRR 5284 transfructosylating enzymes, with a maximum yield of 61%. Intracellular, extracellular, and total A. pullulans FRR 5284 transfructosylating enzymes from different production systems resulted in different FOS profiles, indicating that FOS profiles can be controlled by adjusting intracellular and extracellular enzyme ratios and, hence prebiotic activity.

Transformation of sugarcane molasses into fructooligosaccharides with enhanced prebiotic activity using whole-cell biocatalysts from Aureobasidium pullulans FRR 5284 and an invertase-deficient Saccharomyces cerevisiae 1403-7A.

Fructooligosaccharides (FOS) can be used as feed prebiotics, but are limited by high production costs. In this study, low-cost sugarcane molasses was used to produce whole-cell biocatalysts containing transfructosylating enzymes by Aureobasidium pullulans FRR 5284, followed by FOS production from molasses using the whole-cells of A. pullulans. A. pullulans in molasses-based medium produced cells and broth with a total transfructosylating activity of 123.6 U/mL compared to 61.0 and 85.8 U/mL in synthetic molasses-based and sucrose-based media, respectively. It was found that inclusion of glucose in sucrose medium reduced both transfructosylating and hydrolytic activities of the produced cells and broth. With the use of pure glucose medium, cells and broth had very low levels of transfructosylating activities and hydrolytic activities were not detected. These results indicated that A. pullulans FRR 5284 produced both constitutive and inducible enzymes in sucrose-rich media, such as molasses while it only produced constitutive enzymes in the glucose media. Furthermore, treatment of FOS solutions generated from sucrose-rich solutions using an invertase-deficient Saccharomyces yeast converted glucose to ethanol and acetic acid and improved FOS content in total sugars by 20-30%. Treated FOS derived from molasses improved the in vitro growth of nine probiotic strains by 9-63% compared to a commercial FOS in 12 h incubation. This study demonstrated the potential of using molasses to produce FOS for feed application.

Mild fractionation of sugarcane bagasse into fermentable sugars and ß-O-4 linkage-rich lignin based on acid-catalysed crude glycerol pretreatment.

Acid-catalysed crude glycerol (ACG) pretreatment was carried out at 110 °C and 130 °C for mild fractionation of sugarcane bagasse into fermentable sugars and high-quality lignin. ACG pretreatment at 110 °C led to sugar yields of 71%-74%, comparable to those with acid-catalysed reagent-grade glycerol (AG). ACG pretreatment removed more lignin (53%-75%) than AG pretreatment (38%-49%), likely due to the presence of organic impurities in ACG. Hence, 28% more lignin was recovered from ACG pretreatment hydrolysate than with the AG pretreatment. NMR analysis revealed that recovered lignin was modified by glycerol through etherification of ß-aryl ethers and esterification of hydroxycinnamic acids, which prevented lignin condensation and led to the generation of ß-O-4 linkage-rich lignin at mild conditions (110 °C for 3 h and 5 h). This study suggests that crude glycerol is a suitable low-cost solvent for mild fractionation of lignocellulosic biomass into fermentable sugars and high-quality lignin for value-adding applications.

Scale-up of two-step acid-catalysed glycerol pretreatment for production of oleaginous yeast biomass from sugarcane bagasse by Rhodosporidium toruloides.

Two-step dilute acid and acid-catalysed glycerol pretreatment was developed to maximise sugar yield from sugarcane bagasse. At the laboratory scale, dilute acid pretreatment at 130 °C followed by acid-catalysed glycerol pretreatment at 170 °C led to a total sugar (C5 + C6) yield of 82%, 31% higher than that from one-step acid-catalysed glycerol pretreatment. At the pilot scale, the two-step dilute acid and acid-catalysed glycerol pretreatment led to a maximum sugar yield of 74%, 13% higher than that from one-step pretreatment with 52% reduction in glycerol usage. The enzymatic hydrolysate containing glucose and residual glycerol were used to produce microbial oils by a Rhodosporidium toruloides strain. A fed-batch cultivation strategy led to the production of 44.8 g/L cell mass, including 26.6 g/L oil, 8.6 g/L protein and 12.7 mg/L carotenoid. The cell mass and oil yields were 19% higher than those from batch cultivation as feedstock inhibition and catabolite repression were alleviated.

Co-utilization of acidified glycerol pretreated-sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain.

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co-utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol-pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by-product. It is expected that microbial oil production can be significantly improved through process optimization.

Microbial oil production from acidified glycerol pretreated sugarcane bagasse by Mortierella isabellina.

An integrated microbial oil production process consisting of acidified glycerol pretreatment of sugarcane bagasse, enzymatic hydrolysis, microbial oil production by Mortierella isabellina NRRL 1757 and oil recovery by hydrothermal liquefaction (HTL) of fungal biomass in fermentation broth was assessed in this study. Following pretreatment, the effect of residual pretreatment hydrolysate (containing glycerol) on enzymatic hydrolysis was firstly studied. The residual pretreatment hydrolysate (corresponding to 2.0-7.5% glycerol) improved glucan enzymatic digestibilities by 10-11% compared to the enzymatic hydrolysis in water (no buffer). Although residual pretreatment hydrolysate at 2.0-5.0% glycerol slightly inhibited the consumption of glucose in enzymatic hydrolysate by M. isabellina NRRL 1757, it did not affect microbial oil production due to the consumption of similar amounts of total carbon sources including glycerol. When the cultivation was scaled-up to a 1 L bioreactor, glucose was consumed more rapidly but glycerol assimilation was inhibited. Finally, HTL of fungal biomass in fermentation broth without any catalyst at 340 °C for 60 min efficiently recovered microbial oils from fungal biomass and achieved a bio-oil yield of 78.7% with fatty acids being the dominant oil components (∼89%). HTL also led to the hydrogenation of less saturated fatty acids (C18:2 and C18:3) to more saturated forms (C18:0 and C18:1).

Biofuels from food processing wastes.

Food processing industry generates substantial high organic wastes along with high energy uses. The recovery of food processing wastes as renewable energy sources represents a sustainable option for the substitution of fossil energy, contributing to the transition of food sector towards a low-carbon economy. This article reviews the latest research progress on biofuel production using food processing wastes. While extensive work on laboratory and pilot-scale biosystems for energy production has been reported, this work presents a review of advances in metabolic pathways, key technical issues and bioengineering outcomes in biofuel production from food processing wastes. Research challenges and further prospects associated with the knowledge advances and technology development of biofuel production are discussed.

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 was reduced by 4.5% and 11.0%, respectively. However, glycerol did not irreversibly inhibit cellulase enzymes. Ethanol fermentation was not affected by glycerol up to 5.0 wt%, but was inhibited slightly at 10.0 wt% glycerol, resulting in reduction in ethanol yield from 86.0% in the absence of glycerol to 83.7% (20 h). Based on the results of laboratory and pilot-scale experiments, it was estimated that 0.142 kg ethanol can be produced from 1.0 kg dry bagasse (a glucan content of 38.0%) after pretreatment with acidified glycerol solution.

A multi-criteria analysis approach for ranking and selection of microorganisms for the production of oils for biodiesel production.

Oleaginous microorganisms have potential to be used to produce oils as alternative feedstock for biodiesel production. Microalgae (Chlorella protothecoides and Chlorella zofingiensis), yeasts (Cryptococcus albidus and Rhodotorula mucilaginosa), and fungi (Aspergillus oryzae and Mucor plumbeus) were investigated for their ability to produce oil from glucose, xylose and glycerol. Multi-criteria analysis (MCA) using analytic hierarchy process (AHP) and preference ranking organization method for the enrichment of evaluations (PROMETHEE) with graphical analysis for interactive aid (GAIA), was used to rank and select the preferred microorganisms for oil production for biodiesel application. This was based on a number of criteria viz., oil concentration, content, production rate and yield, substrate consumption rate, fatty acids composition, biomass harvesting and nutrient costs. PROMETHEE selected A. oryzae, M. plumbeus and R. mucilaginosa as the most prospective species for oil production. However, further analysis by GAIA Webs identified A. oryzae and M. plumbeus as the best performing microorganisms.

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

BACKGROUND: 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. RESULTS: 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 110°C to 170°C. 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 k a ,k b and k d . 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, α, was required in the model to ensure reproducibility of the experimental oligomer profiles at 110°C, 125°C and 140°C. The parameters obtained through the fitting exercises at lower temperatures were able to be used to predict the oligomer profiles at 155°C and 170°C with promising results. CONCLUSIONS: 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.

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.

Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols.

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100°C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100°C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100°C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90°C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%) to that obtained in glycerol alone, while Tm-EG retained only 35%. With acidified ethylene glycol under these conditions, both Tl-EG and Tm-EG retained 36% of their activity. The results therefore show that Tl-EG is more stable in both acidified glycerol and ethylene glycol than Tm-EG. A preliminary kinetic study showed that pure glycerol improved the thermal stability of Tl-EG but destabilized Tm-EG, relative to the buffer solution. The half-lives of both Tl-EG and Tm-EG are 4.5 min in acidified glycerol, indicating that the effectiveness of these enzymes under typical pretreatment times of greater than 15 min will be considerably diminished. Attempts have been made to explain the differences in the results obtained between the two enzymes.