Separate Enzymatic Hydrolysis And Fermentation Research Articles

Technologies and factors affecting bioethanol fermentation and its commercialization

Fossil fuels are a major contributor to climate change and environmental pollution, and as the demand for energy production increases, alternative sources are becoming more attractive. Bioethanol reduce reliance on fossil fuels and can be compatible with the existing fleet of internal combustion engines. Bioethanol is typically produced via microbial fermentation of fermentable sugars. Traditional feedstocks (first- generation) include cereal grains, sugar cane, and sugar beets. However, due to concerns regarding food sustainability, lignocellulosic (second-generation) and algal biomass (third-generation) feedstocks have been investigated. Technologies such as Simultaneous Saccharification and Fermentation (SSF), Separate enzymatic Hydrolysis and Fermentation (SHF) and Fed- batch Fermentation for bioethanol hold tremendous potential for the production of bioethanol. The aim of this review focuses on the technologies and factors affecting bioethanol production and its commercialization.

Enhancing the ethanol production by exploiting a novel metagenomic-derived bifunctional xylanase/β-glucosidase enzyme with improved β-glucosidase activity by a nanocellulose carrier.

Some enzymes can catalyze more than one chemical conversion for which they are physiologically specialized. This secondary function, which is called underground, promiscuous, metabolism, or cross activity, is recognized as a valuable feature and has received much attention for developing new catalytic functions in industrial applications. In this study, a novel bifunctional xylanase/β-glucosidase metagenomic-derived enzyme, PersiBGLXyn1, with underground β-glucosidase activity was mined by in-silico screening. Then, the corresponding gene was cloned, expressed and purified. The PersiBGLXyn1 improved the degradation efficiency of organic solvent pretreated coffee residue waste (CRW), and subsequently the production of bioethanol during a separate enzymatic hydrolysis and fermentation (SHF) process. After characterization, the enzyme was immobilized on a nanocellulose (NC) carrier generated from sugar beet pulp (SBP), which remarkably improved the underground activity of the enzyme up to four-fold at 80°C and up to two-fold at pH 4.0 compared to the free one. The immobilized PersiBGLXyn1 demonstrated 12 to 13-fold rise in half-life at 70 and 80°C for its underground activity. The amount of reducing sugar produced from enzymatic saccharification of the CRW was also enhanced from 12.97 g/l to 19.69 g/l by immobilization of the enzyme. Bioethanol production was 29.31 g/l for free enzyme after 72 h fermentation, while the immobilized PersiBGLXyn1 showed 51.47 g/l production titre. Overall, this study presented a cost-effective in-silico metagenomic approach to identify novel bifunctional xylanase/β-glucosidase enzyme with underground β-glucosidase activity. It also demonstrated the improved efficacy of the underground activities of the bifunctional enzyme as a promising alternative for fermentable sugars production and subsequent value-added products.

High Titer Ethanol Production from Combined Alkaline/Alkaline Hydrogen Peroxide Pretreated Bamboo at High Solid Loading

Enzymatic hydrolysis and fermentation at high solid loading is generally required for the production of high titer ethanol from lignocellulosic biomass at a relatively low cost. In this paper, bamboo pretreated by alkaline and alkaline hydrogen peroxide was subjected to downstream enzymatic hydrolysis and ethanol fermentation at high solid loading for the production of high titer ethanol. The effects of solid loading (5–25% w/v) and enzyme dosage (10–50 mg protein/g glucan) on sugar conversion and ethanol production during separate enzymatic hydrolysis and fermentation (SHF) and simultaneous enzymatic saccharification and co-fermentation (SSCF) processes were investigated. During the fermentation process, the pentose-hexose fermenting Saccharomyces cerevisiae LF1 strain was used. The results showed that the increase of solid loading decreased sugar conversion during enzymatic hydrolysis. At relatively high solid loadings (≥ 20% w/v), SSCF resulted in higher sugar conversion and final ethanol titer compared to SHF. With this proposed process, an ethanol concentration of 68.2 g/L, along with approximately 83.5% of glucan and 73.8% of xylan conversions, could be reached through SSCF at 20% solid loading with an enzyme dosage of only 20 mg protein/g glucan.

Bioethanol production from bamboo with alkali-catalyzed liquid hot water pretreatment

Altering recalcitrant structures of bamboo is essential to obtain high yield of bioethanol via bioconversion process. With the goal of improving cell wall digestibility, alkaline liquid hot water was used to pretreat N. affinis. The effects of temperature and alkali dosage on structural alterations were determined by chemical composition, Brunauer Emmett Teller (BET) and gel permeation chromatography (GPC). The relationship between these changes and substrate digestibility was addressed by separate enzymatic hydrolysis and fermentation (SHF). The results indicated that pretreatments partly removed and degraded hemicelluloses and lignin, reducing yields of substrates and molecular weights of carbohydrates. With the change of cell wall structure, specific surface area of materials increased after LHW pretreatment but decreased with further removal of lignin and hemicellulosic fractions. Maximum bioconversion was obtained by pretreatment with 0.5% NaOH aqueous at 170 °C and SHF, yielding 4.8 g/L ethanol.

Improved cellulosic ethanol production from corn stover with a low cellulase input using a β-glucosidase-producing yeast following a dry biorefining process.

A low-cost and sustainable cellulosic ethanol production is vital for fermentation-based industrial applications. Reducing the expenses of cellulose-deconstruction enzymes is one of the significant challenges to economic cellulose-to-ethanol conversion. Here, we report the improved ethanol production from corn stover after dry biorefining using a natural β-glucosidase-producing strain Clavispora NRRL Y-50464 with a low cellulase dose of 5mg protein/g glucan under separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) conditions. Strain Clavispora NRRL Y-50464 exhibited a superior ethanol fermentation performance over Saccharomyces cerevisiae DQ1 under both conditions. It produced an ethanol titer of 38.1g/L within 96h at a conversion efficiency of 55.5% with 25% solids loading (w/w) via SSF without addition of extra β-glucosidase supplement. Improved performance of Y-50464 on a bioreactor with a helical stirring apparatus confirmed its advantage over the conventional bioreactors originally designed for liquid fermentations in cellulosic ethanol conversion by SSF. The results of this study suggested that the strain Clavispora NRRL Y-50464 has a potential as a candidate for lower-cost cellulosic ethanol production from lignocellulosic materials.

Comparative study on hydrolysis and bioethanol production from cardoon and rockrose pretreated by dilute acid hydrolysis

Bioethanol production potential from cardoon (Cynara cardunculus) and rockrose (Cistus ladanifer), pretreated by diluted acid hydrolysis (DAH), was studied and compared by both, separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes. DAH pretreatment allowed an increase of 5 fold to cardoon and 16 fold to rockrose in cellulose accessibility towards enzymatic hydrolysis. Both plants showed better overall fermentation efficiency and ethanol yield in SSF than in SHF also with shorter processing periods, 24 h against 72 + 24 h, respectively. Results obtained for rockrose are about half the concentration and volumetric productivity of ethanol and 60% of the overall fermentation efficiency of those obtained for cardoon. The recalcitrance of rockrose to enzymatic hydrolysis was explained by higher abundance of lignin and extractives than in cardoon. Comparing the steam explosion (SE) and DAH pretreatments, the former was more advantageous for the recalcitrant rockrose in terms of enzymatic saccharification yield.

Investigation of food waste valorization through sequential lactic acid fermentative production and anaerobic digestion of fermentation residues

This work concerns the investigation of the sequential production of lactic acid (LA) and biogas from food waste (FW). LA was produced from FW using a Streptococcus sp. strain via simultaneous saccharification and fermentation (SSF) and separate enzymatic hydrolysis and fermentation (SHF). Via SHF a yield of 0.33gLA/gFW (productivity 3.38gLA/L·h) and via SSF 0.29gLA/gFW (productivity 2.08gLA/L·h) was obtained. Fermentation residues and FW underwent anaerobic digestion (3wt% TS). Biogas yields were 0.71, 0.74 and 0.90Nm3/kgVS for FW and residues from SSF and SHF respectively. The innovation of the approach is considering the conversion of FW into two different products through a biorefinery concept, therefore making economically feasible LA production and valorising its fermentative residues. Finally, a mass balance of three different outlines with the aim to assess the amount of LA and biogas that may be generated within different scenarios is presented.

Bioethanol production from steam explosion pretreated and alkali extracted Cistus ladanifer (rockrose)

Biofuels are suitable alternatives to conventional and fossil fuels that pose serious environmental adverse effects to society. Bioethanol is the most promising biofuel and can be generated from lignocellulosic biomasses. Forest residues are outside the human food chain and inexpensive raw materials, but they need a previous pretreatment, in order to improve the cellulose accessibility for further bioconversion.The study of bioethanol production from rockrose pretreated by steam explosion (SE) was carried out employing separate enzymatic hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) approaches. Saccharification of untreated rockrose attained only 0.9% of sugars yield. However, the steam explosion pretreatment promoted the disruption of interfibrillar surfaces of fibers with partial degradation of lignin thus enhancing the accessibility of polysaccharides toward enzymatic hydrolysis. Alkaline extraction after steam explosion pretreatment of rockrose residue (R-SE-OH) led to the partial removal of lignin, hemicelluloses, and other degradation products from fibre surface allowing an increase of 75% in the glucose yield. Bioethanol production in SSF mode was faster and slightly more efficient process than SHF providing the best results: ethanol concentration of 16.1gL−1, fermentation efficiency of 69.8% and a yield of 22.1g ethanol/100g R-SE-OH.

Recent advances in production of succinic acid from lignocellulosic biomass

Production of succinic acid via separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) are alternatives and are environmentally friendly processes. These processes have attained considerable positions in the industry with their own share of challenges and problems. The high-value succinic acid is extensively used in chemical, food, pharmaceutical, leather and textile industries and can be efficiently produced via several methods. Previously, succinic acid production via chemical synthesis from petrochemical or refined sugar has been the focus of interest of most reviewers. However, these expensive substrates have been recently replaced by alternative sustainable raw materials such as lignocellulosic biomass, which is cheap and abundantly available. Thus, this review focuses on succinic acid production utilizing lignocellulosic material as a potential substrate for SSF and SHF. SSF is an economical single-step process which can be a substitute for SHF - a two-step process where biomass is hydrolyzed in the first step and fermented in the second step. SSF of lignocellulosic biomass under optimum temperature and pH conditions results in the controlled release of sugar and simultaneous conversion into succinic acid by specific microorganisms, reducing reaction time and costs and increasing productivity. In addition, main process parameters which influence SHF and SSF processes such as batch and fed-batch fermentation conditions using different microbial strains are discussed in detail.

Attainable region analysis for continuous production of second generation bioethanol

BackgroundDespite its semi-commercial status, ethanol production from lignocellulosics presents many complexities not yet fully solved. Since the pretreatment stage has been recognized as a complex and yield-determining step, it has been extensively studied. However, economic success of the production process also requires optimization of the biochemical conversion stage. This work addresses the search of bioreactor configurations with improved residence times for continuous enzymatic saccharification and fermentation operations. Instead of analyzing each possible configuration through simulation, we apply graphical methods to optimize the residence time of reactor networks composed of steady-state reactors. Although this can be easily made for processes described by a single kinetic expression, reactions under analysis do not exhibit this feature. Hence, the attainable region method, able to handle multiple species and its reactions, was applied for continuous reactors. Additionally, the effects of the sugars contained in the pretreatment liquor over the enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) were assessed.ResultsWe obtained candidate attainable regions for separate enzymatic hydrolysis and fermentation (SHF) and SSF operations, both fed with pretreated corn stover. Results show that, despite the complexity of the reaction networks and underlying kinetics, the reactor networks that minimize the residence time can be constructed by using plug flow reactors and continuous stirred tank reactors. Regarding the effect of soluble solids in the feed stream to the reactor network, for SHF higher glucose concentration and yield are achieved for enzymatic hydrolysis with washed solids. Similarly, for SSF, higher yields and bioethanol titers are obtained using this substrate.ConclusionsIn this work, we demonstrated the capabilities of the attainable region analysis as a tool to assess the optimal reactor network with minimum residence time applied to the SHF and SSF operations for lignocellulosic ethanol production. The methodology can be readily modified to evaluate other kinetic models of different substrates, enzymes and microorganisms when available. From the obtained results, the most suitable reactor configuration considering residence time and rheological aspects is a continuous stirred tank reactor followed by a plug flow reactor (both in SSF mode) using washed solids as substrate.

Efficient conversion of sugarcane stalks into ethanol employing low temperature alkali pretreatment method

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and β-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12 h, achieving 88% of the theoretical yield from the sugars and cellulose.

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.

Biochemical conversion of rice straw into bioethanol - an exploratory investigation

A good amount of research has been carried in recent years on the production of ethanol from lignocellulosic mass because it is not only a viable and eco-friendly alternative for the fossil fuels but also offers a solution to biomass waste management. Biochemical conversion of cellulosic biomass to ethanol can be done either by simultaneous saccharification and fermentation (SSF) or by separate enzymatic hydrolysis and fermentation (SHF) processes. In the present study, the readily available rice straw was chosen as a lignocellulosic biomass and pretreated with sulphuric acid and ammonium hydroxide to remove lignin for enhancing porosity and hence enzyme permeability. The pretreated straw was characterised by FT-IR and TG studies. Ethanol was produced from the acid pretreated straw by SHF method using acid and cellulase enzyme catalysis successively for saccharification and Baker's yeast (Saccharomyces cerevisiae) for the subsequent fermentation of the obtained sugar. Ethanol produced was estimated spectrophotometrically and compared with the theoretical yield. The greater degree of thermal degradation in the temperature range 200–400°C for the acid pretreated rice straw compared to the virgin sample impliedthe former's decreased structural integrity and improved flexibility. This was corroborated by the enhanced ethanol yield from the SHF process for the combined successive acid and enzymatic saccharification compared to the SHF process involving enzymatic saccharification only. The economic viability of this method has also been discussed in the present study.

Py-GC/MS for characterization of non-hydrolyzed residues from bioethanol production from softwood

Analytical pyrolysis combined with gas chromatography/mass spectrometry (Py-GC/MS) was used to analyze chemical composition of non-hydrolyzed residues (LHRs) obtained by three methods of bioethanol production: softwood acid hydrolysis (AH), separate enzymatic hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF). Complementary techniques, such as EPR- and FTIR-spectroscopy, and routine chemical analysis procedures were used for this study as well. The Py-GC/MS analysis of the LHRs has shown a higher efficiency of carbohydrates hydrolysis upon SSF process in comparison with SHF and AH processes. Comparison of chemical analysis results and data obtained by Py-GC/MS of LHRs brought the direct evidence of incorporation of carbohydrates-derived fragments into the lignin matrix and formation of so-called pseudo-lignin upon different stages of softwood processing. Modifications of lignin component of LHRs on various stages of the process of bioethanol production, such as oxidation and condensation reactions, cleavage of ether bonds and destruction of side propane chain, were revealed using Py-GC/MS.

Influence of High Solid Concentration on Enzymatic Hydrolysis and Fermentation of Steam-Exploded Corn Stover Biomass

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.

Enzyme-based hydrolysis processes for ethanol from lignocellulosic materials: A review

This article reviews developments in the technology for ethanol production from lignocellulosic materials by “enzymatic” processes. Several methods of pretreatment of lignocelluloses are discussed, where the crystalline structure of lignocelluloses is opened up, making them more accessible to the cellulase enzymes. The characteristics of these enzymes and important factors in enzymatic hydrolysis of the cellulose and hemicellulose to cellobiose, glucose, and other sugars are discussed. Different strategies are then described for enzymatic hydrolysis and fermentation, including separate enzymatic hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), non-isothermal simultaneous saccharification and fermentation (NSSF), simultaneous saccharification and co-fermentation (SSCF), and consolidated bioprocessing (CBP). Furthermore, the by-products in ethanol from lignocellulosic materials, wastewater treatment, commercial status, and energy production and integration are reviewed.

Bioethanol production from bio‐ organosolv pulps of Pinus radiata and Acacia dealbata

AbstractWood chips from Pinus radiata and Acacia dealbata were pretreated with the white‐rot fungi Ceriporiopsis subvermispora and Ganoderma australe, respectively, for 30 days at 27 °C and 55% relative humidity, followed by an organosolv delignification with 60% ethanol solution at 200 °C for 1 h to produce pulps with high cellulose and low lignin content. Biotreatment for 30 days was chosen based on low weight and cellulose losses (lower than 4%) and lignin degradation higher than 9%. After organosolv delignification, pulp yield for P. radiata and A. dealbata pulps was 45–49% and 31–51%, respectively. P. radiata bio‐pulps showed higher glucan (93%) and lower lignin content (6%) than control pulps (82% glucan and 13% lignin). A. dealbata bio‐pulps also showed higher glucan (95%) and lower lignin content (2%) than control pulps (92% glucan and 4% lignin). Pulp suspensions at 2% consistency were submitted either to separate enzymatic hydrolysis and fermentation (SHF) or simultaneous enzymatic saccharification and fermentation (SSF) for bioethanol production. The yeast Saccharomyces cerevisiae was used for fermentation. Glucan‐to‐glucose conversion in the enzymatic hydrolysis of control and bio‐pulps of P. radiata was 55% and 100%, respectively, and it was 100% for all pulp samples case of A. dealbata. The highest ethanol yield (calculated as percentage of theoretical yield) during SHF of P. radiata control and bio‐pulps was 38% and 55%, respectively, and for A. dealbata control and bio‐pulps 62% and 69%, respectively. The SSF of P. radiata control and bio‐pulps yielded 10% and 65% of ethanol, respectively, and 77% and 82% for A. dealbata control and bio‐pulps, respectively. In wood basis, the maximum conversion obtained (g ethanol per kg wood) in SHF was 37% and 51% (for P. radiata and A. dealbata pulps, respectively) and 44% and 65% in SSF (for P. radiata and A. dealbata pulps, respectively) regarding the theoretical yield. The low wood‐to‐ethanol conversion was associated with low pulp yield (A. dealbata pulps), high residual lignin amount (P. radiata pulps) and the low pulp consistency (2%) used for SHF and SSF. Copyright © 2007 Society of Chemical Industry