Sulfite pretreatment (SPORL) for robust enzymatic saccharification of spruce and red pine

This study established a novel process using sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust and efficient bioconversion of softwoods. The process consists of sulfite treatment of wood chips under acidic conditions followed by mechanical size reduction using disk refining. The results indicated that after the SPORL pretreatment of spruce chips with 8–10% bisulfite and 1.8–3.7% sulfuric acid on oven dry (od) wood at 180 °C for 30 min, more than 90% cellulose conversion of substrate was achieved with enzyme loading of about 14.6 FPU cellulase plus 22.5 CBU β-glucosidase per gram of od substrate after 48 h hydrolysis. Glucose yield from enzymatic hydrolysis of the substrate per 100 g of untreated od spruce wood (glucan content 43%) was about 37 g (excluding the dissolved glucose during pretreatment). Hemicellulose removal was found to be as critical as lignin sulfonation for cellulose conversion in the SPORL process. Pretreatment altered the wood chips, which reduced electric energy consumption for size reduction to about 19 Wh/kg od untreated wood, or about 19 g glucose/Wh electricity. Furthermore, the SPORL produced low amounts of fermentation inhibitors, hydroxymethyl furfural (HMF) and furfural, of about 5 and 1 mg/g of untreated od wood, respectively. In addition, similar results were achieved when the SPORL was applied to red pine. By building on the mature sulfite pulping and disk refining technologies already practiced in the pulp and paper industry, the SPORL has very few technological barriers and risks for commercialization.     

Enhancement of the enzymatic digestibility of sugarcane bagasse by alkali–peracetic acid pretreatment

The enzymatic digestibility of sugarcane bagasse was greatly increased by alkali (NaOH)–peracetic acid (PAA) pretreatment under mild conditions. The effects of several factors affecting the pretreatment were investigated. It was found that when bagasse was pre-pretreated by 10% (based on initial dry materials) NaOH with 3:1 liquid-to-solid ratio at 90 °C for 1.5 h and further delignified by 10% peracetic acid (based on initial dry materials) at 75 °C for 2.5 h, the yield of reducing sugars reached 92.04% by enzymatic hydrolysis for 120 h with cellulase loading of 15 FPU/g solid. Compared with acid and alkali pretreatment, alkali–PAA pretreatment could be conducted under milder conditions and was more effective for delignification with less carbohydrates being degraded in the pretreatment process. Alkaline stage played an important role for partial delignification, swelling fibers and subsequently reducing PAA loading. No loss of cellulase activity (FPA) was observed in the liquid phase for alkali–PAA pretreated bagasse after enzymatic hydrolysis for 120 h.     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

Statistical optimization of enzymatic degradation process for oil palm empty fruit bunch (OPEFB) in rotary drum bioreactor using crude cellulase produced from Aspergillus niger EFB1

Oil palm empty fruit bunch (OPEFB) was pretreated with 2% (v/v) HNO₃ and degraded by Aspergillus niger EFB1 crude cellulase. Through 2 Level Factorial Design (2LFD), it was found that OPEFB concentration, temperature, incubation time, concentration of Tween 80 and agitation speed have significant effect in reducing sugar production. A standard Response Surface Methodology (RSM) design known as Central Composite Design (CCD) was used to optimize the enzymatic degradation condition of OPEFB in rotary drum bioreactor. Reducing sugar level of 1.183 g/L was obtained with the following optimized degradation conditions: 1.95% (w/v) OPEFB, 0.5% (v/v) Tween 80, 55 °C, 87.5 rpm in the incubation period of 3 days and 16 h. The optimal degradation condition improved reducing sugar production by 1.07 fold compared to that before optimization in shake flasks culture. The optimization strategy of enzymatic degradation of OPEFB inside rotary drum bioreactor led to increase in glucose, xylose, arabinose, galactose and mannose production by 3, 2.5, 1.64, 19.37 and 22.52 fold, respectively. The improvement in reducing sugar and polyoses production were comparable with the reduction in OPEFB cellulose and hemicellulose content by 89.32% and 48.17% respectively after enzymatic degradation in optimized condition.     

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g⁻¹, while in the fermentation carried out with a cell concentration of 2 g L⁻¹ a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L⁻¹ h⁻¹ were achieved.     

Pretreatment of sugarcane bagasse using supercritical carbon dioxide combined with ultrasound to improve the enzymatic hydrolysis

This work evaluates the pretreatment of sugarcane bagasse combining supercritical carbon dioxide (SC-CO₂) and ultrasound to enhance the enzymatic hydrolysis of pretreated bagasse. In a first step the influence of process variables on the SC-CO₂ pretreatment to enhance the enzymatic hydrolysis was evaluated by mean of a Plackett–Burmann design. Then, the sequential treatment combining ultrasound + SC-CO₂ was evaluated. Results show that treatment using SC-CO₂ increased the amount of fermentable sugar obtained of about 280% compared with the non-treated bagasse, leading to a hydrolysis efficiency (based on the amount of cellulose) as high as 74.2%. Combining ultrasound + SC-CO₂ treatment increased about 16% the amount of fermentable sugar obtained by enzymatic hydrolysis in comparison with the treatment using only ultrasound. From the results presented in this work it can be concluded that the combined ultrasound + SC-CO₂ treatment is an efficient and promising alternative to carry out the pretreatment of lignocellulosic feedstock at relatively low temperatures without the use of hazardous solvents.     

Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose

β-Glucosidase immobilized on magnetic chitosan microspheres for potential recycling usage in hydrolysis of cellulosic biomass was investigated. The immobilized enzyme had an activity of 6.4 U/g support under optimized condition when using cellobiose as substrate. Immobilization resulted in less increase of the apparent K_m, low drift of the optimal pH, as well as improved stability relative to the free enzyme. The immobilized β-glucosidase was applied to enzymatic hydrolysis of corn straw to produce 60.2 g/l reducing sugar with a conversion rate of 78.2% over the course of a 32-h reaction. This conversion rate was maintained above 76.5% after recycling the enzyme for use in eight batches (total 256 h), showing favorable operational stability of the immobilized enzyme.     

Subcritical water and dilute acid pretreatments for bioethanol production from Melaleuca leucadendron shedding bark

The feasibility of bioethanol production using the lignocellulose of the shedding bark of Melaleuca leucadendron (Paper bark tree) was investigated. The effects of pretreatment parameters (temperature, time and acid concentration) on the yields of sugars and inhibitors, and optimal pretreatment conditions were determined. At very low severity conditions (combined severity factor, CSF ≤ 0.335), 28% of xylan was recovered and this recovery increased with increasing CSF till it peaked to 64.4% (11.2 g xylose L⁻¹) at a CSF of 1.475. However, at CSF > 2.0, xylose yield declined due to degradation. Mild and progressive glucose yield was detected in prehydrolysate at CSF ≥ 1.514, and subsequent enzymatic hydrolysis allowed complete glucan solubilization. Implementing environmentally friendly subcritical water pretreatment at CSF ≤ 0.335 on the shedding bark, about 85% of glucan solubilization was achieved after enzymatic hydrolysis. An industrial Saccharomyces cerevisiae strain readily fermented crude hydrolysate within 12 h, yielding 24.7 g L⁻¹ ethanol at an inoculum size of 2% (v/v), representing a glucose to ethanol conversion rate of 0.475 g g⁻¹ (91% ethanol yield). Based on our findings, the shedding bark is a potential feedstock for bio-ethanol production.     

Evaluation of the cold-active Pseudoalteromonas haloplanktis β-galactosidase enzyme for lactose hydrolysis in whey permeate as primary step of d-tagatose production

d-Tagatose is an innovative natural low-calorie bulk sweetener with a broad potential for low-calorie and low-glycaemic foods and drinks. Production of this healthy sweetener is realized through enzymatic d-galactose isomerization. d-Galactose needs to be produced in situ due to its limited availability. Whey permeate contains a substantial amount of lactose, which is an interesting source for d-galactose production through enzymatic lactose hydrolysis. In this context, the cold-active β-galactosidase from the psychrophile Pseudoalteromonas haloplanktis was studied. Optimal parameters for efficient lactose hydrolysis in whey permeate have been deduced, viz. optimal incubation temperature, pH and lactose concentration. Hydrolysis efficiencies above 96.0% were realized within 24 h at 23 °C and pH 7.0 in whey permeate with a maximum dry matter content of 10.0% (w/w). In addition, the effect of the presence of d-glucose and d-galactose was investigated up to concentrations of 100 g l⁻¹. d-Glucose inhibited lactose hydrolysis more strongly compared to d-galactose. Also, the operational stability of the cold-active β-galactosidase was studied. Hydrolysis efficiencies above 90.0% were maintained during 7 subsequent hydrolysis cycles.     

High-loading oil palm empty fruit bunch saccharification using cellulases from Trichoderma koningii MF6

Strain Trichoderma koningii D-64 was improved for enhanced cellulase production. A potential mutant MF6 was obtained and its enzymes contained filter paper cellulase (FPase), carboxymethylcellulase (CMCase), β-glucosidase and xylanase with respective activities of 2.0, 1.3, 2.0 and 3.0 folds of those for the parental strain. MF6 cellulases showed enhanced hydrolysis performance for the treated lignocellulosic biomass. Hydrolysis of treated oil palm empty fruit bunch (OPEFB), horticulture wastes (HW) and wood chips (WC) resulted in cellulose to glucose conversion of 96.3 ± 2.2%, 98.2 ± 3.0% and 81.9 ± 1.4%, respectively. The corresponding conversions of xylan to xylose were 96.9 ± 1.5%, 95.0 ± 2.2% and 76.1 ± 3.1%. Consistently, high sugar yield of 770–844 mg/g biomass was obtained for high-loading (10–16%, w/v) of OPEFB hydrolysis and sugar titer of 135.1 g/L was obtained for 16% (w/v) OPEFB loading at 96 h. In addition, MF6 enzymes alone performed equally well for high-loading OPEFB hydrolysis compared to the enzyme mixture of β-glucosidase from Aspergillus niger and cellulase from T. reesei Rut C30.     

Comparisons of SPORL and dilute acid pretreatments for sugar and ethanol productions from aspen

This study reports comparative evaluations of sugar and ethanol production from a native aspen (Populus tremuloides) between sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) and dilute acid (DA) pretreatments. All aqueous pretreatments were carried out in a laboratory wood pulping digester using wood chips at 170°C with a liquid to oven dry (od) wood ratio (L/W) of 3:1 at two levels of acid charge on wood of 0.56 and 1.11%. Sodium bisulfite charge on od wood was 0 for DA and 1.5 or 3.0% for SPORL. All substrates produced by both pretreatments (except DA with pretreatment duration of 0) had good enzymatic digestibility of over 80%. However, SPORL produced higher enzymatic digestibility than its corresponding DA pretreatment for all the experiments conducted. As a result, SPORL produced higher ethanol yield from simultaneous saccharification and fermentation of cellulosic substrate than its corresponding DA pretreatment. SPORL was more effective than its corresponding DA pretreatment in reducing energy consumption for postpretreatment wood chip size-reduction. SPORL, with lower energy input and higher sugar and ethanol yield, produced higher sugar and ethanol production energy efficiencies than the corresponding DA pretreatment.     

Sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust enzymatic saccharification of hardwoods

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of hardwoods. With only about 4% sodium bisulfite charge on aspen and 30‐min pretreatment at temperature 180°C, SPORL can achieve near‐complete cellulose conversion to glucose in a wide range of pretreatment liquor of pH 2.0–4.5 in only about 10 h enzymatic hydrolysis. The enzyme loading was about 20 FPU cellulase plus 30 CBU β‐glucosidase per gram of cellulose. The production of fermentation inhibitor furfural was less than 20 mg/g of aspen wood at pH 4.5. With pH 4.5, SPORL avoided reactor corrosion problem and eliminated the need for substrate neutralization prior to enzymatic hydrolysis. Similar results were obtained from maple and eucalyptus. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Hemicellulose sugar recovery from steam-exploded wheat straw for microbial oil production

There are currently few successful examples of using straw hemicellulose as a carbon source in the fermentation industry. In this paper, hemicellulose hydrolysates were recovered from steam-exploded wheat straw (SEWS) and used to produce microbial oil. The effects of the steam explosion treatment conditions, the elution temperature and the ratio of elution water to SEWS on sugar recovery were examined. A broth with 3.8 g l^?1 of reducing sugar and 22.3 g l^?1 of total soluble sugars was obtained with a 10-fold excess (w/w) of water at 40 °C to wash the SEWS treated under steam explosion conditions at 200 °C for 5 min. This broth was used to produce microbial oil by the oleaginous fungus Microsphaeropsis sp., which was able to secrete xylanase to degrade oligosaccharides from straw hemicellulose and accumulate microbial oil. Under optimized conditions, the oil concentration was 2.6 g l^?1. The yield of oil from sugar consumed was 0.14 g g^?1. The microbial oil produced by this research could be used as feedstock for biodiesel production because the microbial oil was primarily composed of neutral lipids. This research establishes a novel protocol for microbial oil production from straw hemicellulose.     

Comparison of Dilute Acid and Sulfite Pretreatment for Enzymatic Saccharification of Earlywood and Latewood of Douglas fir

This study applied dilute acid (DA) and sulfite pretreatment to overcome the recalcitrance of lignocelluloses (SPORL) to deconstruct earlywood and latewood cell walls of Douglas fir for fermentable sugars production through subsequent enzymatic hydrolysis. DA pretreatment removed almost all the hemicelluloses, while SPORL at initial pH?=?4.5 (SP-B) removed significant amount of lignin between 20 and 25 %. But both are not sufficient for effective enzymatic saccharification. SPORL at low initial pH?=?2 (SP-AB) combines the advantage of both DA and SPORL-B to achieve approximately 90 % hemicellulose removal and delignification of 10–20 %. As a result, SP-AB effectively removed recalcitrance and thereby significantly improved enzymatic saccharification compared with DA and SP-B. Results also showed that earlywood with significantly lower density produced less saccharification after DA pretreatment, suggesting that wood density does not contribute to recalcitrance. The thick cell wall of latewood did not limit chemical penetration in pretreatments. The high lignin content of earlywood limited the effectiveness of DA pretreatment for enzymatic saccharification, while hemicellulose limits the effectiveness of high pH pretreatment of SP-B. The higher hemicellulose content in the earlywood and latewood of heartwood reduced saccharification relative to the corresponding earlywood and latewood in the sapwood using DA and SP-AB.     

Two-step acid and alkaline ethanolysis/alkaline peroxide fractionation of sugarcane bagasse and rice straw for production of polylactic acid precursor

Sugarcane bagasse and rice straw were subjected to acid and alkaline ethanolysis and sequential enzymatic hydrolysis to produce glucose for lactic acid production. Influence of physico-chemical treatments using ultrasonic bath and ultrasonic probe was studied compared with mechanical stirring. The results showed that the highest glucose yield with least contamination of xylose was obtained from acid ethanolysis fractionation (5 N H₂SO₄ + 50%, v/v ethanol) when stirred at 90 °C for 4 h. Alkaline ethanolysis accomplished high amount of both glucose and xylose released, however it was not favorable substrate for homofermentative lactic acid bacteria. In order to enhance enzymatic hydrolysis of acid ethanolysis fractionated samples, lignin was subsequently removed by the second step alkaline/peroxide delignification. The maximum lactic acid was obtained at 23.6 ± 0.2 g/L from Lactobacillus casei fermentation after 72 h when hydrolysate from two-step acid hydrolysis and alkaline/peroxide fractionated sugarcane bagasse containing 24.6 g/L initial glucose concentration was used as substrate.     

Enhanced enzymatic hydrolysis of hydrothermally pretreated empty fruit bunches at high solids loadings by the synergism of hemicellulase and polyethylene glycol

In enzymatic hydrolysis, high lignocellulose loadings are required to obtain high sugar titers. However, the high solids loadings limit enzymatic hydrolysis. In this study, to overcome this limitation, the promoting and synergistic effects of the accessory agents of hemicellulase (i.e., Cellic HTec2) and polyethylene glycol (PEG) 8000 were investigated in the enzymatic hydrolysis of hydrothermally pretreated empty fruit bunches (EFBs). After the optimal addition of Cellic HTec2 and PEG, high enzymatic digestion of the pretreated EFBs was achieved owing to their synergistic effects, even at high solids loadings. For example, the enzymatic digestibility of pretreated EFBs at a 21.7% (w/v) solids loading with 10 FPU of Cellic CTec2/g glucan reached 72.5% when 2.7 mg of Cellic HTec2/g glucan and 62.5 mg of PEG/g glucan were used as the accessory agents. These results suggested that the optimal addition of accessory agents is effective for the enhanced hydrolysis of lignocellulose using even a commercial cellulase preparation.     

Effect of inoculum and pepsin–pancreatin hydrolysis on fibre fermentation measured by the gas production technique in pigs

Two experiments were undertaken to adapt the in vitro gas production technique in syringes, used for ruminants, to fibre fermentation studies in the large intestine of pigs.In a first experiment, two inocula (faeces and large intestine content) were compared at four dilution levels in a buffer solution (0.025, 0.05, 0.1 and 0.2 g ml⁻¹) with two substrates: wheat bran and sugar–beet pulp. The accumulated gas produced over 72 h was modelled and the kinetics parameters compared. The time to half asymptote was lower for the intestinal inoculum (5.5 versus 8.0 h, P<0.02), but the 2 inocula yielded similar fractional rates of degradation (0.16 h⁻¹) and gave equal final gas production (252 ml g⁻¹ substrate). No interaction (P>0.05) was observed between inocula and substrates. The dilution of the samples in the buffer solution increased (P<0.001) the lag time (from 0.9 to 2.1 h for dilution rates ranging from 0.2 to 0.025 g ml⁻¹, respectively) and decreased (P<0.001) the rates of substrate degradation (from 0.18 to 0.13 h⁻¹).A second experiment aimed to study the effect of an in vitro pepsin–pancreatin hydrolysis of the sample prior to the gas test. Six substrates were tested: maize, wheat bran, sugar–beet pulp, lupins, peas and soybean meal. The enzymatic hydrolysis affected (P<0.001) the kinetics parameters and the ranking order of the fermented substrates. The lag times also increased for all ingredients. The rate of degradation decreased when peas, lupins, maize and wheat bran were hydrolysed (P<0.001) but it increased with soybean meal (P=0.014) and sugar–beet pulp (P<0.001). Final gas production increased with peas and soybean meal (P<0.001), remained unchanged for lupins and decreased for the other substrates (P<0.001).In conclusion, the method using faeces as a source of microbial inoculum is reliable to characterise the fermentation kinetics of ingredients in the large intestine of pigs. However, it is important to hydrolyse the substrates with pepsin and pancreatin before the gas tests.     

Digestive capacities allow the Mexican long-nosed bat (Leptonycteris nivalis) to live in cold environments

Digestive capabilities of nectar-feeding vertebrates to assimilate sugars affect their ability to acquire and store energy and could determine the minimal temperatures at which these animals can survive. Here, we described the sugar digestive capability of Leptonycteris nivalis and related it with its capacity to live in cold environments. We measured the enzymatic activity, food intake rate and changes in body mass of bats feeding at four different sucrose concentrations (from 5 to 35% wt./vol.). Additionally, we used a mathematical model to predict food intake and compared it with the food intake of bats. L. nivalis was able to obtain ~ 111.3 kJ of energy regardless of the sugar concentration of their food. Also, bats gained ~ 2.57 g of mass during the experimental trials and this gain was independent of sugar concentration. The affinity (1 / K_m) of sucrase (EC 3.2.1.48) was one order of magnitude higher relative to that reported for its sister species Leptonycteris yerbabuenae (0.250 and 0.0189 mmol^? 1 L, respectively), allowing this species to have a higher energy intake rate. We propose that the high ability to acquire energy conferred L. nivalis the faculty to invade cold environments, avoiding in this way the ecological competition with its sympatric species L. yerbabuenae.     

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are ³⁴k = 0.994 ± 0.004, ¹³k = 1.0148 ± 0.0007, and ¹⁸k = 0.999 ± 0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.     

Itaconic acid production from wheat chaff by Aspergillus terreus

Biotechnologically produced itaconic acid is an important building block for the chemical industry and still based on pure carbon sources, detoxified molasses or starch hydrolysates. Changing these first generation feedstocks to alternative renewable resources of a second generation implies new challenges for the cultivation process of the industrial itaconic acid producer Aspergillus terreus, which is known to be very sensitive towards impurities. To select a suitable pretreatment method of a second generation feedstock, the influences of different hydrolysate components, like monosaccharides and sugar degradation products, were tested. Particular the impact of those components on itaconic acid yield, productivity, titer and morphology was investigated in detail. Wheat chaff was used as lignocellulosic biomass, which is an agricultural residue. An alkaline pretreatment method with sodium hydroxide at room temperature and a subsequent enzymatic saccharification at pH 4.8 at 50 °C with 10 FPU/g_Biomass Biogazyme 2x proved to be very suitable for a subsequent biotechnological production of itaconic acid. A purification by a cation exchanger of the wheat chaff hydrolysate resulted in a final titer of 27.7 g/L itaconic acid with a yield of 0.41 g/g_{total sugar}.