Characterization of cell wall structure in dilute acid-pretreated biomass by confocal Raman microscopy and enzymatic hydrolysis

The chemical and ultrastructural properties of cell walls were investigated to determine the effect of dilute acid pretreatment on the hydrolysis of lignocellulosic biomass. Confocal Raman microscopy was used to gain a clear understanding of how dilute acid pretreatments destroy lignocellulosic cell walls. Total fermentable sugar (glucose and xylose) was high in oxalic acid hydrolysate (26.18 g/L) compared to that in sulfuric acid hydrolysate (24.34 g/L). Chemical composition of the pretreated biomass differed slightly according to the acid catalyst used. Oxalic acid pretreatment was effective for enzymatic hydrolysis, with 29.46 g/L of total fermentable sugar after 96 h. Optical microscopy showed that dilute acid pretreatment significantly changed cell wall structure, and broken and crushed cell walls could be clearly seen during pretreatment. Based on confocal Raman peak intensity, the ratio of lignin/cellulose [I(1600)/I(900)] was low for oxalic acid-pretreated biomass compared to sulfuric acid-pretreated biomass.     

The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale

The pretreatment of agricultural biomass by diluted acid is often employed to facilitate the release of monosaccharide for the subsequent enzyme hydrolysis for lignocellulosic ethanol production. However, furfural and hydroxymethylfurfural are usually generated and markedly decrease the yield of pentose fermentation during this pretreatment. In the present study, the enhancement of lignocellulosic ethanol production was successfully demonstrated at pilot scale with extra addition of hydrolyzed rice straw into pentose fermentation by Pichia stiptis. This way has resulted into the increase of P. stiptis cell mass was shown to play a positive role. The ethanol yield, 0.45 g_p/g_s, with the addition of hydrolyzed rice straw in hemicellulosic hydrolysate from plywood, bagasse and bamboo were increase 20–51% to demonstrate the applicability of this technology in a variety of lignocellulosic ethanol processes due to the efficient conversion of xylose.     

Material balance studies for the conversion of sorghum stover to bioethanol

The conversion of lignocellulosic biomass to ethanol involves three major unit operations such as pretreatment, hydrolysis and fermentation. Each unit operation involves processing of biomass with changes in its structure, and release of fermentable and other sugars and lignin degrading compounds. The evaluation of biomass conversion processes through material balance is fundamentally crucial in its commercialization. This gives an idea about the transfer of biomass from one phase to another and hence eventually of the efficiency of the total process. In the present study, material balance has been evaluated in each unit operations for sorghum biomass to ethanol conversion. An account of carbohydrates in the native as well as pretreated sorghum biomass, the release of fermentable sugars and the conversion of sugars to ethanol was maintained and analysed. Ethanol yield of 91.94 g per kg sorghum was achieved without any detoxification and washing of pretreated biomass after mild acid pretreatment followed by enzymatic hydrolysis and fermentation.     

Steam pretreatment of dry and ensiled industrial hemp for ethanol production

Biomass can be converted into liquid and gaseous biofuels with good efficiency. In this study, the conversion of industrial hemp (Cannabis sativa L.), a biomass source that can be cultivated with a high biomass yield per hectare, was used. Steam pretreatment of dry and ensiled hemp was investigated prior to ethanol production. The pretreatment efficiency was evaluated in terms of sugar recovery and polysaccharide conversion in the enzymatic hydrolysis step. For both materials, impregnation with 2% SO₂ followed by steam pretreatment at 210 °C for 5 min were found to be the optimal conditions leading to the highest overall yield of glucose. Simultaneous saccharification and fermentation experiments carried out with optimised pretreatment conditions resulted in ethanol yields of 163 g kg^?1 ensiled hemp (dry matter) (71% of the theoretical maximum) and 171 g kg^?1 dry hemp (74%), which corresponds to 206–216 l Mg^?1 ethanol based on initial dry material.     

Effect of sodium sulfite on acid pretreatment of wheat straw with respect to its final conversion to ethanol

A pretreatment process that combines dilute acid and sodium sulfite has been applied to wheat straw to study the effect of temperature (120–180 °C) and sodium sulfite concentration (0–3%) on the yield of glucose in subsequent enzymatic hydrolysis and ethanol production by fermentation. The results were compared with both dilute acid pretreatment (without Na₂SO₃ addition) and hot water pretreatment. Formation of furfural and hydroxymethylfurural, which can inhibit ethanol-producing microorganisms, were measured and the ethanol yield in a subsequent fermentation was evaluated. The results indicate that a combination of 180 °C, 30 min, 1% H₂SO₄ and 2.4% Na₂SO₃ during pretreatment produced the highest ethanol yield; 17.3 g/100 g dry weight of initial biomass, which corresponds to 75% of the theoretical yield from glucose. 28 mg of furan inhibitors (sum of furfural and hydroxymethylfurfural) per gram dry weight of initial wheat straw were generated under this condition. Increasing sulfite loading up to 2.4% decreased inhibitor formation, leading to increased delignification and preservation of cellulose from dissolution. On the other hand, an elevated temperature in combination with low pH reduced the amount of solid phase after pretreatment, increased the level of inhibitors and reduced the concentration of ethanol produced by fermentation.     

Influence of impregnation with lactic acid on sugar yields from steam pretreatment of sugarcane bagasse and spruce, for bioethanol production

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source produced through fermentation of sugars. However, in order to achieve high sugar and ethanol yields, the lignocellulosic material must be pretreated before the enzymatic hydrolysis and fermentation. Dilute acid pretreatment, using SO₂, is one of the most promising methods of pretreatment for softwood and agricultural residues. However, handling the high acidity of the slurry obtained from pretreatment and difficulty in recycling/degradation of the impregnating agent are some of the drawbacks of the dilute acid processes. In the present study the influence of utilization of a weak organic acid (lactic acid), as impregnating agent, on the sugar yield from pretreatment, with and without addition of SO₂, was investigated. The efficiency of pretreatment was assessed by enzymatic hydrolysis of the slurry obtained by pretreatment, using sugarcane bagasse and spruce, stored for one and two months in the presence of lactic acid separately, as feedstocks. Pretreatment of bagasse after storage with 0.5% lactic acid resulted in an overall glucose yield, i.e. after enzymatic hydrolysis, of 79% of theoretical based on the amount available in the raw material. This was as good as pretreatment using SO₂ as impregnating agent. However, storage of spruce with lactic acid before pretreatment, with and without addition of SO₂, was not efficient and resulted in lower sugar yields than pretreatment using SO₂ only.     

Nitrogen explosion pretreatment of lignocellulosic material for bioethanol production

A novel method for the pretreatment of lignocellulosic material is investigated in this work, using floodplain meadow hay as a feedstock for bioethanol production. Pressurized nitrogen (N₂) pretreatment is combined with explosive decompression to achieve high glucose yields with simple technology and low energy input. Results show that N₂ explosion yields hydrolysis efficiencies up to 71.8%. The highest hydrolysis efficiency was achieved at a temperature of 210°C with a cellulose to glucose conversion rate of 195.1 g kg^?1 of biomass.     

Ethanol production from enzymatic hydrolysis of sugarcane bagasse pretreated with lime and alkaline hydrogen peroxide

In this work we evaluated ethanol production from enzymatic hydrolysis of sugarcane bagasse. Two pretreatments agents, lime and alkaline hydrogen peroxide, were compared in their performance to improve the susceptibility of bagasse to enzymatic action. Mild conditions of temperature, pressure and absence of acids were chosen to diminish costs and to avoid sugars degradation and consequent inhibitors formation. The bagasse was used as it comes from the sugar/ethanol industries, without grinding or sieving, and hydrolysis was performed with low enzymes loading (3.50 FPU g⁻¹ dry pretreated biomass of cellulase and 1.00 CBU g⁻¹ dry pretreated biomass of ??-glucosidase). The pretreatment with alkaline hydrogen peroxide led to the higher glucose yield: 691 mg g⁻¹ of glucose for pretreated bagasse after hydrolysis of bagasse pretreated for 1 h at 25 °C with 7.35% (v/v) of peroxide. Fermentation of the hydrolyzates from the two pretreatments were carried out and compared with fermentation of a glucose solution. Ethanol yields from the hydrolyzates were similar to that obtained by fermentation of the glucose solution. Although the preliminary results obtained in this work are promising for both pretreatments considered, reflecting their potential for application, further studies, considering higher biomass concentrations and economic aspects should be performed before extending the conclusions to an industrial process.     

Construction and demolition lignocellulosic wastes to bioethanol

This work deals with conversion of four construction and demolition (C&D) lignocellulosic wastes including OSB, chipboard, plywood, and wallpaper to ethanol by separate enzymatic hydrolysis and fermentation (SHF). Similar to other lignocelluloses, the wastes were resistant to the enzymatic hydrolysis, in which only up to 7% of their cellulose was hydrolyzed. Therefore, the lignocellulosic wastes were treated with phosphoric acid, sodium hydroxide, or N-methylmorpholine-N-oxide (NMMO), which resulted in improving the subsequent enzymatic hydrolysis to 38.2–94.6% of the theoretical yield. The best performance was obtained after pretreatment by concentrated phosphoric acid, followed by NMMO. The pretreated and hydrolyzed C&D wastes were then successfully fermented by baker’s yeast to ethanol with 70.5–84.2% of the theoretical yields. The results indicate the possibility of producing 160 ml ethanol from each kg of the C&D wastes at the best conditions.     

Screening of steam explosion conditions for glucose production from non-impregnated wheat straw

The conversion of wheat straw to fermentable sugar for bioethanol production typically involves a thermal pretreatment step, followed by enzymatic hydrolysis. In this study we have investigated the effect of steam explosion parameters on wheat straw digestibility using a newly designed steam explosion unit and a process without acid impregnation. The wheat straw was steam pretreated using 18 different conditions in the temperature range of 170-220 °C and the resulting material was used directly (i.e. without washing) for enzymatic hydrolysis and fermentation in either a separate hydrolysis and fermentation (SHF)-type or a simultaneous saccharification and fermentation (SSF)-type set-up. Maximum glucose yields upon enzymatic hydrolysis were obtained after pretreatment at 210 °C for 10 min and yields were similar at harsher conditions. Xylose yields increased with temperature and residence time up to 190 °C, but decreased at harsher pretreatment conditions since these led to xylan degradation and concomitant production of furfural. In an SHF-type set-up ethanol formation did not follow enzymatic glucose release and was inversely correlated with furfural levels. An SFF-type set-up displayed a straightforward correlation between the expected amount of released glucose and the ethanol yields. The highest saccharification yields corresponded to about 90% of the cellulose in the substrate. Overall, this study shows that steam explosion without an acid catalyst is a good pretreatment method for saccharification of wheat straw. Optimal steam explosion conditions need to be a compromise between sugar accessibility and sugar degradation.     

Production of anhydrous ethanol using oil palm empty fruit bunch in a pilot plant

Bioethanol production from lignocellulosic biomass for use as an alternative energy resource has attracted increasing interest, but short-term commercialization will require several technologies such as low cost feedstock. The huge amount of oil palm empty fruit bunches (EFB) generated from palm oil industries can be used as a raw material for cheap, renewable feedstock for further commercial exploitation. Using a pilot-scale bioethanol plant, this study investigated the possibility of utilizing oil palm empty fruit bunches as a renewable resource. All bioethanol production processes such as pretreatment, hydrolysis, fermentation, and purification were constructed as automatically controlled integrated processes. The mass balance was calculated from operational results. Changhae ethanol multiexplosion pretreatment with sodium hydroxide was conducted to improve the enzymatic hydrolysis process, and a separate hydrolysis and fermentation process was used for producing bioethanol at an 83.6% ethanol conversion rate. In order to purify the ethanol, a distillation and dehydration facility was operated. Distillation and dehydration efficiencies were 98.9% and 99.2%, respectively. The material balance could be calculated using results obtained from the operation of the pilot-scale bioethanol plant. As a result, it was possible to produce 144.4 kg anhydrous ethanol (99.7 wt%) from 1000 kg EFB. This result constitutes a significant contribution to the feasibility of bioethanol production from lignocellulosic biomass and justifies the pilot plant's scale-up to a commercial-scale plant.     

Effect of enzyme addition on fermentative hydrogen production from wheat straw

Wheat straw is an abundant agricultural residue which can be used as raw material to produce hydrogen (H₂), a promising alternative energy carrier, at a low cost. Bioconversion of lignocellulosic biomass to produce H₂ usually involves three main operations: pretreatment, hydrolysis and fermentation. In this study, the efficiency of exogenous enzyme addition on fermentative H₂ production from wheat straw was evaluated using mixed-cultures in two experimental systems: a one-stage system (direct enzyme addition) and a two-stage system (enzymatic hydrolysis prior to dark fermentation). H₂ production from untreated wheat straw ranged from 5.18 to 10.52 mL-H₂ g-VS⁻¹. Whatever the experimental enzyme addition procedure, a two-fold increase in H₂ production yields ranging from 11.06 to 19.63 mL-H₂ g-VS⁻¹ was observed after enzymatic treatment of the wheat straw. The high variability in H₂ yields in the two step process was explained by the consumption of free sugars by indigenous wheat straw microorganisms during enzymatic hydrolysis. The direct addition of exogenous enzymes in the one-stage dark fermentation stage proved to be the best way of significantly improving H₂ production from lignocellulosic biomass. Finally, the optimal dose of enzyme mixture added to the wheat straw was evaluated between 1 and 5 mg-protein g-raw wheat straw⁻¹.     

Pretreatment of energy crops with sodium hydroxide and cellulolytic enzymes to increase biogas production

This work presents the influence of alkali pretreatment on the enzymatic hydrolysis and efficiency of anaerobic digestion of lignocellulosic biomass pretreated both in a one- (chemical or enzymatic) and two-step (chemical and enzymatic) process. In this study two species of energy crops were used Miscanthus giganteus and Sida hermaphrodita. The aim of this work was to compare biogas production and methane yield during fermentation of pretreated and untreated energy crops. The results show that alkali pretreatment is necessary for the effective biogas generation from plant material due to high delignification level and significant hemicellulose degradation. The two-step hydrolysis process consisting on the alkali and enzymatic step leads to the release of high concentrations of glucose (about 20 g L⁻¹). The best results were achieved for M. giganteus with biogas production yield of 421.5 Ndm³ kg TS⁻¹ and with methane production yield of 257 Ndm³ kg TS⁻¹.     

Ethanol and biogas production from birch by NMMO pretreatment

Birch wood was pretreated with N-methylmorpholine-N-oxide (NMMO or NMO) followed by enzymatic hydrolysis and fermentation to ethanol or digestion to biogas. The pretreatments were carried out with NMMO (w_NMMO = 85%) at 130 °C for 3 h, and the effects of drying after the pretreatment were investigated. Enzymatic hydrolysis of the untreated wood resulted in 8%–10% of theoretical glucose yield after 4 days hydrolysis, while the NMMO pretreatment improved this yield to 91%. Consequently, ethanol production yield from NMMO-pretreated materials resulted in around 9-fold improvement compared to the untreated wood. On the other hand, drying of the pretreated wood had a negative impact and decreased the yield of enzymatic hydrolysis by 4%–10%. Digestion of the untreated wood with thermophilic bacteria resulted in maximum methane yield of 158 cm³ g⁻¹ of VS in 30 days, while the NMMO pretreatment improved the methane yield up to 232 cm³ g⁻¹ of VS (80% of the theoretical biogas yield) in just 9 days.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

Technology for conversion of lignocellulosic biomass to ethanol

Current trends in production of fuel ethanol from lignocellulosic materials are reviewed. Particular emphasis has been laid on the microbial synthesis of cellulases, enzymatic hydrolysis, pretreatment of lignocellulosics, and their simultaneous saccharification and fermentation to ethyl alcohol. Some pilot-scale plants producing alcohol from biomass are also presented.     

Enhanced solid-liquid clarification of lignocellulosic slurries using polyelectrolyte flocculating agents

Following pretreatment and enzymatic hydrolysis of lignocellulosic biomass within a biorefinery, the residual solids are often present in high concentration and have a wide particle size distribution. These solids, which can be difficult to remove from solution, can have a detrimental impact on downstream fermentations and separations. Here we show that polyelectrolyte flocculating agents can be used to enhance the solid-liquid clarification of a lignocellulosic biomass (Ponderosa Pine) hydrolyzate. Due to the negative zeta potential of Ponderosa Pine solids following pretreatment and enzymatic hydrolysis, flocculation with cationic polymers provided the largest flocs, which quickly settled out of solution. Adding Kemira flocculant C1592 up to 1000 mg/L created larger flocs; however, a higher dosage (5000 mg/L) resulted in solids re-dispersing into solution. It was estimated that a greater than 12-fold improvement in throughput with a scroll decanting centrifuge could be obtained when using flocculant, or that an approximately 40-fold higher flux could be obtained for a vacuum filtration operation. The addition of 100 mg/L of C1592 showed optimal suspended solids removal of the clarified solution (<0.1% w/w suspended solids) and the highest sugar recovery (up to 83% without washing). In all cases, the percentage of suspended solids remaining following either centrifugation or filtration was at least an order of magnitude lower with the aid of flocculant. The presence of residual polymer in solution did not affect the downstream ethanol productivity of a Saccharomyces cerevisiae fermentation.     

Sugarcane hybrids with original low lignin contents and high field productivity are useful to reach high glucose yields from bagasse

Five sugarcane hybrids plus a reference material were evaluated according to the glucose yields obtained after alkaline-sulfite pretreatment and enzymatic hydrolysis. Sugarcane hybrids with varied original chemical compositions were used to assess how contrasting samples might influence the integrated pretreatment and hydrolysis process. The hydrolysis efficiency of six samples treated at three different chemical loads, suggested that lignin and hemicellulose removals during the pretreatment were not the single factor necessary to reach high cellulose conversion levels in the enzymatic hydrolysis step. Pretreated samples with the highest total acid contents (mainly sulfonic acids) were also the most digestible materials. The glucose yields were heavily dependent not only on the digestibility of the pretreated materials but also on the field productivity of the plants. One of the hybrids, presenting high glucan yields after pretreatment and high digestibility, produced low glucose yields because it presented very low biomass productivity. In contrast, one of the hybrids that presented low glucan yield after pretreatment, but was highly digestible and presented high biomass productivity, provided the highest glucose yields in the data set, producing 4192 and 5629 kg of glucose per hectare after enzymatic hydrolysis for 24 h and 72 h, respectively.