Simultaneous Enzymatic Saccharification Research Articles

Enhanced reducing sugar production and extraction for Chlorella vulgaris in mixotrophic cultivation using high hydrostatic pressure processing and ultrasound

ABSTRACT Although extraction of polysaccharides to convert reducing sugars (RS) from microalgae by acid or alkali pretreatments and enzymatic hydrolysis has been extensively studied, few reports exploring the use of high hydrostatic pressure processing (HHP) and ultrasonication (US) as emerging technologies for the extraction of sugars from microalgae biomass exist. Thus, the present study was conducted to determine the effects of mixotrophic growth and stress conditions (NaNO3 and CO2 concentration and light intensity) on RS and protein accumulation in the unicellular green alga Chlorella vulgaris in addition to optimization of the effectiveness of the sequential applications of HHP and US with dilute acid as well as simultaneous enzymatic saccharification on the production of RS from microalga cells. High light intensity, high CO2 concentration and limited nitrogen concentration promoted RS production. The maximum protein content (0.0683 mg g‒1) was achieved at 0.3 g l‒1 NaNO3 concentration, 7000 μmol photons m‒2 s‒1 and 6 l min‒1 CO2 concentration. The highest RS content of C. vulgaris after 48 h enzymatic saccharification (583.86 ± 13.23 mg g‒1) was obtained at 1% (w/w) acid concentration and 80% amplitude for 30 min with 79.4% RS yield. Combined US-assisted dilute acid pretreatment and enzymatic hydrolysis were also found to be more effective than HHP assisted dilute acid pretreatment and enzymatic saccharification. Therefore, microalgal biomass can be considered a suitable renewable feedstock used in fermentation.

Electrically conducting films prepared from graphite and lignin in pure water.

In this study, we present electrically conducting self-standing graphite films consisting of lignin derivatives extracted by simultaneous enzymatic saccharification and comminution (SESC). Sonication of graphite powder in the presence of SESC lignin and pure water allows dispersion of the SESC-lignin-attached graphite without addition of other chemicals. The SESC-lignin-attached graphite having a diameter of several micrometers can be used as a surface electroconductive coating and molded into self-standing films by drying. The SESC-lignin-attached graphite film exhibits higher conductivity (∼2,075 S/cm) than graphite-based composites consisting of ordinary lignin derivatives. Manufacturing self-standing films of micrometer-sized graphite using SESC lignin enables high electrical conductivity of the SESC-lignin-attached graphite film. The size of the SESC-lignin-attached graphite is proportional to the conductivity of the film. The SESC-lignin-attached graphite also acts as an antiplasticizer and a conductive filler for polymer films, i.e., conductive films consisting of poly(ethylene glycol) or Li+ montmorillonite can be obtained through a water-based process.

Bubble column bioreactor design and evaluation for bioethanol production using simultaneous saccharification and fermentation strategy from hydrothermally pretreated lignocellulosic biomass

Lignocellulosic biomass serves as an alternative renewable feed stock for the production of bioethanol using biochemical platform. Ethanol yield can be improved by adopting strategies such as simultaneous enzymatic saccharification and fermentation (SSF) using pneumatic bioreactors, robust yeast and biomass pretreated with hydrothermal process. In the present study two strains of Saccharomyces cerevisiae: flocculant (CA11) and non- flocculant (PE-2) were evaluated to grow at temperature. The S. cerevisiae PE-2 was selected for fermentation at 40 ºC for ethanol production and a bubble column bioreactor (BCB) of 3 L was designed and operated for bioethanol production. Hydrothermally pretreated (180 ºC for 30 min) wheat straw rich in cellulose fraction (49.83 g/100 of biomass) with a severity factor (log [Ro] = 4.04) was used as the substrate for SSF strategy for bioethanol production using in a three-phase (gas–liquid–solid) bubble column bioreactor. The ethanol concentration was 9.31 g/L using 10% (w/v) of pretreated solid loading and 15 FPU/g substrate of enzyme loading in a BCB. The robust yeasts, pneumatic bioreactors and hydrothermal pretreatment are a great alternative in the production of biofuels at an industrial level in terms of second generation biorefineries.

Improvement of saccharide yield from wood by simultaneous enzymatic delignification and saccharification using a ligninolytic enzyme and cellulase

White-rot fungi are thought to hold promise for development of a delignification pretreatment process for wood biorefinery that is less energy-consuming than current processes. However, the reaction must take place over weeks and consumes non-neglectable amounts of saccharides. To establish a biological process for wood biorefinery would first require establishment of an enzymatic approach to delignification. Such an approach has the potential to lower costs and reduce saccharide loss. Here, we attempted enzymatic delignification reactions using manganese peroxidases (MnP), a lignin-degrading enzyme, under several reaction conditions. The delignification rate from beech wood meal (particle size <45μm) of up to 11.0% in 48h was reached in a MnP reaction supplemented with multiple co-oxidants, glucose, glucose oxidase (GOD) and commercial cellulase. An additional 48-h reaction using fresh MnP/co-oxidants increased the delignification rate to 14.2%. Simultaneous enzymatic delignification and saccharification, which occurs without a need for glucose supplementation, successfully improved the glucose yield to 160% of the reaction without MnP. Development of a more accurate imitation of the mechanisms of delignification that occurs in white-rot fungi has the potential to improve the monosaccharide yield resulting from simultaneous delignification and saccharification.

Polymer heatproofing mechanism of lignin extracted by simultaneous enzymatic saccharification and comminution

In this paper, we reveal a synthetic polymer heatproofing mechanism of a lignin derivative extracted by simultaneous enzymatic saccharification and comminution (hereafter referred to as “SESC lignin”). First, the prevention of back-biting on the thermal degradation of poly(ethylene carbonate) by SESC lignin is experimentally verified via chromatograph/spectroscopic approaches. Second, we explore the applicable scope (e.g., the limiting temperature) of SESC lignin as a heatproof filler by comparing the heatproof properties of a conventional heat stabilizer and SESC lignin. Finally, we confirm that the combination of “preferential pyrolysis” and “radical scavenging” of SESC lignin induces its synthetic polymer heatproof properties, which can be determined using thermochemical/kinetical approaches.

Functional Materials from Plant Biomass Obtained by Simultaneous Enzymatic Saccharification and Communition

Functional Materials from Plant Biomass Obtained by Simultaneous Enzymatic Saccharification and Communition

Functional Materials from Plant Biomass Obtained by Simultaneous Enzymatic Saccharification and Communition

Functional Materials from Plant Biomass Obtained by Simultaneous Enzymatic Saccharification and Communition

Strong UV absorption by nanoparticulated lignin in polymer films with reinforcement of mechanical properties

In this paper, we report about function of nanoparticulated lignin derivative as a UV absorber for polymer films. The lignin derivative obtained by simultaneous enzymatic saccharification and comminution (SESC lignin) exhibited strong UV absorption in a poly(vinyl alcohol) matrix without dramatically affecting its transparency. Furthermore, a small amount of SESC lignin simultaneously acted as a mechanical/thermal enhancer of the polymer film. The presented UV absorption system does not require toxic additives; i.e., the SESC lignin is expected for utilization as a UV absorber in food/medicine packaging films. Furthermore, the SESC lignin can be prepared via an environmentally friendly process. Therefore, the strong UV absorption property of the SESC lignin not only promotes the utilization of plant biomass as a high-value industrial material but also reduces the environmental load caused by heatproof materials derived from toxic petroleum-based resources.

Combined simultaneous enzymatic saccharification and comminution (SESC) and anaerobic digestion for sustainable biomethane generation from wood lignocellulose and the biochemical characterization of residual sludge solid

Simultaneous enzymatic saccharification and comminution (SESC) was used for large-scale anaerobic digestion of wood lignocellulose to generate methane and unmodified lignin. During SESC, 10% aqueous mixture of powdered debarked wood from various species was subjected to bead milling with hydrolytic enzymes to generate particles below 1 μm. This slurry was directly used as a cosubstrate for anaerobic digestion in a 500 L stirred-tank reactor. Temperature and hydraulic retention time (HRT) were maintained at 50 °C and 30 days, respectively. At stable operation periods, an average yield of 224 L of methane per kg of cedar was attained. Comparable yields were achieved with red pine, elm, oak, and cedar bark. High-throughput microbial analysis established the presence of a relevant community to support the elevated level of methane production. The stability of the unmodified lignin in anaerobic digestion was also confirmed, allowing for its recovery as an important by-product.

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.

Simultaneous enzymatic saccharification and comminution for the valorization of lignocellulosic biomass toward natural products

BackgroundLarge-scale processing of lignocellulosics for glucose production generally relies on high temperature and acidic or alkaline conditions. However, extreme conditions produce chemical contaminants that complicate downstream processing. A method that mainly rely on mechanical and enzymatic reaction completely averts such problem and generates unmodified lignin. Products from this process could find novel applications in the chemicals, feed and food industry. But a large-scale system suitable for this purpose is yet to be developed. In this study we applied simultaneous enzymatic saccharification and communition (SESC) for the pre-treatment of a representative lignocellulosic biomass, cedar softwood, under both laboratory and large-scale conditions.ResultsLaboratory-scale comminution achieved a maximum saccharification efficiency of 80% at the optimum pH of 6. It was possible to recycle the supernatant to concentrate the glucose without affecting the efficiency. During the direct alcohol fermentation of SESC slurry, a high yield of ethanol was attained. The mild reaction conditions prevented the generation of undesired chemical inhibitors. Large-scale SESC treatment using a commercial beads mill system achieved a saccharification efficiency of 60% at an energy consumption of 50 MJ/kg biomass.ConclusionSESC is very promising for the mild and clean processing of lignocellulose to generate glucose and unmodified lignin in a large scale. Economic feasibility is highly dependent on its potential to generate high value natural products for energy, specialty chemicals, feed and food application.

Production of various organic acids from different renewable sources by immobilized cells in the regimes of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SFF)

The study was aimed at production of different organic acids (OA) (lactic, fumaric, or succinic) by various microbial cells (filamentous fungi Rhizopus oryzae (F-814, F-1127) and bacteria Actinobacillus succinogenes B-10111) immobilized into poly(vinyl alcohol) (PVA) cryogel from diverse renewable raw materials (wheat and rice straw, aspen and pine sawdust, Jerusalem artichoke stems and tubers, biomass of macro- and microalgae) under batch conditions. The process productivity, bulk output and OA concentrations were higher in case of using immobilized cells than in case of free cells under identical conditions. A higher OA productivity was reached via simultaneous enzymatic saccharification and microbial fermentation (SSF) of same raw materials as compared to their separate enzymatic hydrolysis and fermentation of accumulated reducing sugars (SHF). Maximal concentrations of all OAs studied were obtained for bioconversion of Jerusalem artichoke tubers. The immobilized cells were used in long-term conversion of various renewable materials to OAs in SSF.

Plant-Based Antioxidant Nanoparticles without Biological Toxicity.

Here, we present a function to derive non‐deteriorated nanoparticulated lignin as an antioxidant without biological toxicity that is supplied through the simultaneous enzymatic saccharification and comminution of plants. The lignin exhibits an oxygen radical absorption capacity, even in its macromolecular nature. The non‐deteriorated lignin nanoparticles never inhibit the biological activity of living things, despite their antioxidant nature. The oxygen radical absorption capacity of lignin is dependent on its botanical origin and monomeric structure. A stable organic radical in lignin is responsible for the antioxidant nature of non‐deteriorated lignin. The organic radical of non‐deteriorated lignin, which yields a distinct signal on electron spin resonance spectra, serves as a spin trap reagent that detects the emergence of short lifespan radicals as the change of radical concentration of the lignin. The presented discovery of non‐deteriorated lignin will induce not only the industrial utilization of plant biomass polymers in pharmaceuticals and reagents, but also advance our scientific understanding of the antioxidant function of native lignin.

Utilization of Lignocellulosic Biomass via Novel Sustainable Process.

In this review, we show novel methods for utilizing lignocellulosic biomass, polysaccharides, and lignin. Firstly, the simultaneous enzymatic saccharification and comminution (SESC) of plant materials is described as an extraction method for lignocellulosic biomass that does not require toxic reagents or organic solvents. Secondly, we demonstrate the material utilization of non-deteriorated lignocellulosic biomass extracted by SESC, such as for sugar and ethanol synthesis, and as a heatproof filler. Finally, we exhibit the use of a functional monomer (e.g., in disinfection chemicals, cesium chelation, and building blocks for polymers), 2-pyrone-4,6-dicarboxylic acid, derived from lignin via metabolic degradation.

Microbial production of mannitol by Lactobacillus brevis 3-A5 from concentrated extract of Jerusalem artichoke tubers.

In the present study, the conversion of the extract of Jerusalem artichoke tubers for mannitol production by Lactobacillus brevis 3-A5 was investigated. When the bacterium utilized enzymatic hydrolysates of Jerusalem artichoke extract as the main substrates in batch fermentation, the significant decrease in mannitol productivity was observed when the initial concentration of reducing sugar increased. Then, a strategy of continuous fed-batch fermentation was adopted for improving mannitol production with enzymatic hydrolysates of Jerusalem artichoke extract as main substrates. Although the concentration of mannitol could reach 199.86 g/L at the end of the fermentation, the productivity for the overall process of the fermentation was only 1.67 g/L/H. To improve the mannitol productivity with both higher yield and concentration, the simultaneous enzymatic saccharification and fermentation (SSF) was studied. In SSF, the mannitol production reached 176.50 g/L in 28 H with a productivity of 6.30 g/L/H and a yield of 0.68 g/g total sugar. Our study provides a cost-effective and eco-friendly method for mannitol production from a cheap biomass.

Complex effect of lignocellulosic biomass pretreatment with 1-butyl-3-methylimidazolium chloride ionic liquid on various aspects of ethanol and fumaric acid production by immobilized cells within SSF

The pretreatment of softwood and hardwood samples (spruce and hornbeam wood) with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was undertaken for further simultaneous enzymatic saccharification of renewable non-food lignocellulosic biomass and microbial fermentation of obtained sugars to ethanol and fumaric acid. A multienzyme cocktail based on cellulases and yeast or fungus cells producing ethanol and fumaric acid were the main objects of [Bmim]Cl influence studies. A complex effect of lignocellulosic biomass pretreatment with [Bmim]Cl on various aspects of the process (both action of cellulases and microbial conversion of hydrolysates to target products) was revealed. Positive effects of the pretreatment with [Bmim]Cl included decreasing the lignin content in the biomass, and increasing the effectiveness of enzymatic hydrolysis and microbial transformation of pretreated biomass. Immobilized cells of both yeasts and fungi possessed improved productive characteristics in the biotransformation of biomass pretreated with [Bmim]Cl to ethanol and fumaric acid.

Optimization of simultaneous saccharification and fermentation conditions with amphipathic lignin derivatives for concentrated bioethanol production

Amphipathic lignin derivatives (A-LDs) prepared from the black liquor of soda pulping of Japanese cedar are strong accelerators for bioethanol production under a fed-batch simultaneous enzymatic saccharification and fermentation (SSF) process. To improve the bioethanol production concentration, conditions such as reaction temperature, stirring program, and A-LDs loadings were optimized in both small scale and large scale fed-batch SSF. The fed-batch SSF in the presence of 3.0g/L A-LDs at 38°C gave the maximum ethanol production and a high enzyme recovery rate. Furthermore, a jar-fermenter equipped with a powerful mechanical stirrer was designed for 1.5L-scale fed-batch SSF to achieve rigorous mixing during high substrate loading. Finally, the 1.5L fed-batch SSF with a substrate loading of 30% (w/v) produced a high ethanol concentration of 87.9g/L in the presence of A-LDs under optimized conditions.

Acetic acid-catalyzed hydrothermal pretreatment of corn stover for the production of bioethanol at high-solids content.

Corn stover (CS) was hydrothermally pretreated using CH3COOH (0.3%, v/v), and subsequently its ability to be utilized for conversion to ethanol at high-solids content was investigated. Pretreatment conditions were optimized employing a response surface methodology (RSM) with temperature and duration as independent variables. Pretreated CS underwent a liquefaction/saccharification step at a custom designed free-fall mixer at 50°C for either 12 or 24h using an enzyme loading of 9mg/g dry matter (DM) at 24% (w/w) DM. Simultaneous enzymatic saccharification and fermentation (SSF) of liquefacted corn stover resulted in high ethanol concentration (up to 36.8g/L), with liquefaction duration having a negligible effect. The threshold of ethanol concentration of 4% (w/w), which is required to reduce the cost of ethanol distillation, was surpassed by the addition of extra enzymes at the start up of SSF achieving this way ethanol titer of 41.5g/L.

Ethanol Production from Microwave-assisted FeCl3 Pretreated Rice Straw

Microwave-assisted FeCl3 pretreated rice straw was used for enzymatic saccharification and ethanol production. Reducing sugars concentration from pretreated rice straw was 5.85 g/L after 48 h, which was higher than that from untreated rice straw. Simultaneous enzymatic saccharification and fermentation of untreated and pretreated rice straw was carried out using Saccharomyces cerevisiae, Pichia stipitis, Saccharomyces cerevisiae and Pichia stipitis, and immobilized Saccharomyces cerevisiae and Pichia stipitis cells. Ethanol production from pretreated rice straw had shorter reaction time, and could achieve higher ethanol concentration than untreated rice straw in these simultaneous enzymatic saccharification and fermentation processes. Ethanol concentration from simultaneous enzymatic saccharification and fermentation with immobilized Saccharomyces cerevisiae and Pichia stipitis cells was higher than that from simultaneous enzymatic saccharification and fermentation with Saccharomyces cerevisiae, simultaneous enzymatic saccharification and fermentation with Pichia stipites, and simultaneous enzymatic saccharification and fermentation with Saccharomyces cerevisiae and Pichia stipitis. The maximum ethanol concentration obtained from simultaneous enzymatic saccharification and fermentation of pretreated rice straw using immobilized Saccharomyces cerevisiae and Pichia stipitis cells was 5.51 g/L after 48 h.

Non-sterile simultaneous saccharification and fermentation of corn leaves and stalks to l-lactic acid without external nutrient addition

Fermentation of lignocellulosic biomass requires auxiliary materials, including nutrients, to ensure the proliferation of microorganisms. Nutrients are usually inexpensive, but their contribution to the cost is considerable because of the very low prices of fermentation products, such as bio-ethanol. Using substances present in native lignocellulosic biomass as nutrients for fermentation was proposed and demonstrated. Leaves and stalks of corn plants were used as biomass, and nutrients were recovered as a nutrient solution by soaking them in water before alkaline peroxide pretreatment. Pretreated biomass and the nutrient solution derived from the same lot were used for non-sterile simultaneous enzymatic saccharification and thermophilic l-lactic acid fermentation (SSF). Using the nutrient solution in the saccharification step did not impact sugar recovery, and instead improved sugar yields because of the presence of eluted sugars in the solution. The l-lactic acid yield of 0.33 g g−1 based on native biomass weight indicated that the nutrient solution functioned as a source of nutrients and sugars, especially as a source of essential phosphorus. Comparatively, autoclaved SSF yielded less or no l-lactic acid, indicating an apparent inhibitive effect derived from the nutrient solution on bacterial growth.