Untreated Lignocellulosic Biomass Research Articles

Cross-linked enzyme aggregates of xylanase, XynR8(N58D), for effective degradation of untreated lignocellulosic biomass

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process.

A Comparison of Dilute Aqueous Isethionic Acid and Sulfuric Acid in Hydrolysis of Three Different Untreated Lignocellulosic Biomass Varieties.

Efficient catalytic hydrolysis of lignocellulosic biomass to sugars is a major challenge in the production of sustainable biofuels and chemical feedstocks. In this study isethionic acid was compared with H2SO4 for hydrolysis of polysaccharides in corn stover, switch grass, and poplar. The catalytic activities of acids were compared by analysis of total reducing sugar (TRS) and glucose yields in a sequence of experiments in water at 90-190 °C using 0.050 mol of H+/L isethionic acid and H2SO4. In comparison to using H2SO4, the use of isethionic acid catalyst lowered the maximum TRS percent yield temperatures by 25, 24, and 21% for corn stover, switch grass, and poplar. A similar effect was observed for glucose percent yields as well. This temperature reduction is due to lowering of the activation energy in the polysaccharide depolymerization reaction and most likely due to hydrogen-bonding-type dipolar interactions between the isethionic acid -OH group and -OH groups in biomass polysaccharides.

Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides.

Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 carbohydrate-binding module, two fibronectin type-3 domains, and a family 3 carbohydrate-binding module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli. Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite - 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications. KEY POINTS: • PcAA10A is a novel modular LPMO family 10 from Paenibacillus curdlanolyticus. • PcAA10A showed broad substrate specificity on β-1,4 glycosidic linkage substrates. • Non-catalytic domains are important for degrading complex polysaccharides. • PcAA10A is a unique LPMO capable of enhancing lignocellulosic biomass degradation.

The extracellular endo-\u03b2-1,4-xylanase with multidomain from the extreme thermophile Caldicellulosiruptor lactoaceticus is specific for insoluble xylan degradation

BackgroundThe extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus can degrade and metabolize untreated lignocellulosic biomass containing xylan. The mechanism of the bacterium for degradation of insoluble xylan in untreated biomass has not been revealed.ResultsIn the present study, the only annotated extracellular endo-β-1,4-xylanase (Xyn10B) with multidomain structures in C. lactoaceticus genome was biochemically characterized. Xyn10B contains three N-terminal consecutive family 22 carbohydrate-binding modules (CBMs), one GH10 catalytic domain (CD), two family 9 CBMs and two S-layer homology (SLH) modules in the C-terminal. CBM22a shares 27.1% and 27.2% sequence homology with CBM22b and CBM22c, respectively. The sequence homology between two CBM9 s and two SLHs is 26.8% and 25.6%, respectively. To elucidate the effect of multiple domains on the enzymatic properties of Xyn10B, the truncated variants of which (Xyn10B-TM1: CBM22a-CBM22b-CBM22c-CD10; Xyn10B-TM2: CBM22c-CD10; Xyn10B-TM3: CBM22c-CD10-CBM9a; and Xyn10B-TM4: CD10-CBM9a) were separately reconstructed, recombinantly expressed and biochemically characterized. Enzymatic properties studies showed that the optimal temperature for all four Xyn10B truncations was 65 °C. Compared to Xyn10B-TM3 and Xyn10B-TM4, Xyn10B-TM1 and Xyn10B-TM2 had higher hydrolytic activity, thermostability and affinity on insoluble substrates. It is noteworthy that Xyn10B-TM1 and Xyn10B-TM2 have higher enzymatic activity on insoluble xylan than the soluble counterparts, whereas Xyn10B-TM3 and Xyn10B-TM4 showed opposite characteristics. The kinetic parameters analysis of Xyn10B-TM1 on xylan showed Vmax was 5740, 1300, 1033, and 3925 U/μmol on insoluble oat spelt xylan (OSX), soluble beechwood xylan (BWX), soluble sugar cane xylan (SCX), and soluble corncob xylan (CCX), respectively. The results indicated that CBM22s especially CBM22c promoted the hydrolytic activity, thermostability and affinity on insoluble substrates of the Xyn10B truncations. The functions of CBM22, CBM9, CD and SLH are different, while contribute synergetically to the thermostability, protein structure integrity, substrate binding, and high hydrolytic activity on insoluble xylan of untreated lignocellulosic biomass. The domains of CBM22, CBM9, CD and SLH have different characteristics, which synergistically promote the thermostability, protein structure integrity, affinity on insoluble substrates and enzymatic activity properties of Xyn10B.ConclusionsThe extracellular endo-β-1,4-xylanase with multidomain structures of CBM, CD and SLH promote the biodegradation of insoluble xylan in untreated lignocellulosic biomass by thermophilic C. lactoaceticus.

Thermostable Xylanase Production by Geobacillus sp. Strain DUSELR13, and Its Application in Ethanol Production with Lignocellulosic Biomass

The aim of the current study was to optimize the production of xylanase, and its application for ethanol production using the lignocellulosic biomass. A highly thermostable crude xylanase was obtained from the Geobacillus sp. strain DUSELR13 isolated from the deep biosphere of Homestake gold mine, Lead, SD. Geobacillus sp. strain DUSELR13 produced 6 U/mL of the xylanase with the beechwood xylan. The xylanase production was improved following the optimization studies, with one factor at a time approach, from 6 U/mL to 19.8 U/mL with xylan. The statistical optimization with response surface methodology further increased the production to 31 U/mL. The characterization studies revealed that the crude xylanase complex had an optimum pH of 7.0, with a broad pH range of 5.0–9.0, and an optimum temperature of 75 °C. The ~45 kDa xylanase protein was highly thermostable with t1/2 of 48, 38, and 13 days at 50, 60, and 70 °C, respectively. The xylanase activity increased with the addition of Cu+2, Zn+2, K+, and Fe+2 at 1 mM concentration, and Ca+2, Zn+2, Mg+2, and Na+ at 10 mM concentration. The comparative analysis of the crude xylanase against its commercial counterpart Novozymes Cellic HTec and Dupont, Accellerase XY, showed that it performed better at higher temperature, hydrolyzing 65.4% of the beechwood at 75 °C. The DUSEL R13 showed the mettle to hydrolyze, and utilize the pretreated, and untreated lignocellulosic biomass: prairie cord grass (PCG), and corn stover (CS) as the substrate, and gave a maximum yield of 20.5 U/mL with the untreated PCG. When grown in co-culture with Geobacillus thermoglucosidasius, it produced 3.53 and 3.72 g/L ethanol, respectively with PCG, and CS. With these characteristics the xylanase under study could be an industrial success for the high temperature bioprocesses.

Renewable Resources and a Recycled Polymer as Raw Materials: Mats from Electrospinning of Lignocellulosic Biomass and PET Solutions.

Interest in the use of renewable raw materials in the preparation of materials has been growing uninterruptedly in recent decades. The aim of this strategy is to offer alternatives to the use of fossil fuel-based raw materials and to meet the demand for materials that are less detrimental to the environment after disposal. In this context, several studies have been carried out on the use of lignocellulosic biomass and its main components (cellulose, hemicelluloses, and lignin) as raw materials for polymeric materials. Lignocellulosic fibers have a high content of cellulose, but there has been a notable lack of investigations on application of the electrospinning technique for solutions prepared from raw lignocellulosic biomass, even though the presence of cellulose favors the alignment of the fiber chains during electrospinning. In this investigation, ultrathin (submicrometric) and nanoscale aligned fibers were successfully prepared via electrospinning (room temperature) of solutions prepared with different contents of lignocellulosic sisal fibers combined with recycled poly(ethylene terephthalate) (PET) using trifluoroacetic acid (TFA) as solvent. The “macro” fibers were deconstructed by the action of TFA, resulting in solutions containing their constituents, i.e., cellulose, hemicelluloses, and lignin, in addition to PET. The “macro” sisal fibers were reconstructed at the nanometer and submicrometric scale from these solutions. The SEM micrographs of the mats containing the components of sisal showed distinct fiber networks, likely due to differences in the solubility of these components in TFA and in their dielectric constants. The mechanical properties of the mats (dynamic mechanical analysis, DMA, and tensile properties) were evaluated with the samples positioned both in the direction (dir) of and in opposition (op) to the alignment of the nano and ultrathin fibers, which can be considered a novelty in the analysis of this type of material. DMA showed superior values of storage modulus (E’ at 30 °C) for the mats characterized in the preferential direction of fiber alignment. For example, for mats obtained from solutions prepared from a 0.4 ratio of sisal fibers/PET, Sisal/PET0.40dir presented a high E’ value of 765 MPa compared to Sisal/PET0.40op that presented an E’ value of 88.4 MPa. The fiber alignment did not influence the Tg values (from tan δ peak) of electrospun mats with the same compositions, as they presented similar values for this property. The tensile properties of the electrospun mats were significantly impacted by the alignment of the fibers: e.g., Sisal/PET0.40dir presented a high tensile strength value of 15.72 MPa, and Sisal/PET0.40op presented a value of approximately 2.5 MPa. An opposite trend was observed regarding the values of elongation at break for these materials. Other properties of the mats are also discussed; such as the index of fiber alignment, average porosity, and surface contact angle. To our knowledge, this is the first time that the influence of fiber alignment on the properties of electrospun mats based on untreated lignocellulosic biomass combined with a recycled polymer, such as PET, has been evaluated. The mats obtained in this study have potential for diversified applications, such as reinforcement for polymeric matrices in nanocomposites, membranes for filtration, and support for enzymes, wherein the fiber alignment, together with other evaluated properties, can impact their effectiveness in these applications.

Coproduction of protease and amylase by thermophilic Bacillus sp. BBXS-2 using open solid-state fermentation of lignocellulosic biomass

•Untreated lignocellulosic biomass is fermented to produce bacterial enzymes.•An open (nonaseptic) solid-state fermentation is used to minimize costs.•Thermophilic/alkalophilic fermentation conditions could control contamination.•Much higher titers of enzymes were produced compared to the literature.•Enzymes added to laundry detergents improved stain removal by ∼2.5-fold.

A novel approach for extracting cellulose nanofibers from lignocellulosic biomass by ball milling combined with chemical treatment

ABSTRACTCellulose nanofibers (CNFs) were isolated from kenaf fibers and wheat straw by formic acid (FA)/acetic acid (AA), peroxyformic acid (PFA)/peroxyacetic acid (PAA), hydrogen peroxide (H2O2) treatment; and subsequently through ball milling treatment. Characterization of extracted cellulose and cellulose nanofibers was carried out through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD), and thermogravimetric analysis (TGA). TEM images showed that extracted cellulose nanofibers had diameter in the range of 8–100 nm. FTIR and XRD results implied that hemicellulose and lignin were mostly removed from lignocellulosic biomass with an increase in crystallinity, and isolation of cellulose nanofibers was successful. The TGA results showed that decomposition temperature of cellulose nanofibers increased by about 27°C when compared with that of untreated lignocellulosic biomass. No significant change was observed in the decomposition temperature of bleached celluloses after ball milling. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 42990.

Bioconversion of un-pretreated lignocellulosic materials by a microbial consortium XDC-2

The present study investigated the degradation of un-pretreated wheat straw, corn stalk, and rice straw by a lignocellulose-degrading microbial consortium XDC-2. Following six days of cultivation, exocellular xylanase activities were 414.9, 491.9, and 335U/mL, respectively. After 12days, the rice straw had lost 39.71% of its weight, hemicellulose and cellulose losses of 78.27% and 14.08%, respectively. The total amount of volatile products reached a maximum on day six for rice straw degradation. The four major types of volatile products were acetic acid, propionic acid, butanoic acid, and glycerin, all of which would be suitable substrates for conversion to methanol by anaerobic digestion. According to PCR-DGGE analysis, XDC-2 remained stable during the degradation process of untreated lignocellulosic biomass. These results demonstrate the potential for further development and application of XDC-2; it is capable of degrading un-pretreated lignocellulosic materials, and has a low cost of operation.

Conversion of xylan, d-xylose and lignocellulosic biomass into furfural using AlCl3 as catalyst in ionic liquid

In order to define a new green catalytic pathway for the production of furfural, the catalyzed conversion of xylan into furfural in 1-butyl-3-methylimidazolium chloride was studied by using mineral acids and metal chlorides as catalysts under microwave irradiation. Amongst these catalysts, AlCl3 resulted in the highest furfural yield of 84.8% at 170°C for 10s. The effect of AlCl3 on the conversion efficiency of d-xylose and untreated lignocellulosic biomass was also investigated, the yields of furfural from corncob, grass and pine wood catalyzed by AlCl3 in [BMIM]Cl were in the range of 16–33%. [BMIM]Cl and AlCl3 could be recycled for four runs with stable catalytic activity. AlCl3 is less corrosive than mineral acids, and the use of ionic liquid as reaction medium will no longer generate toxic wastewater, thus this reaction system is more ecologically viable.

COMPUTER-ASSISTED ANALYSIS OF FOURIER TRANSFORM INFRARED (FTIR) SPECTRA FOR CHARACTERIZATION OF VARIOUS TREATED AND UNTREATED AGRICULTURE BIOMASS

A computational approach was used to analyze the FTIR spectra of a wide range of treated and untreated lignocellulosic biomass (coconut husk, banana trunk, sago hampas, rice husk, and empty fruit bunch). The biomass was treated with strong sulphuric acid and NaOH, respectively. A total of 87 spectra were obtained in which the absorption bands were de-convoluted automatically, generating a peak table of 87 rows and 60 columns. Square roots were taken of the peak values, with further standardization prior to Principal Component Analysis (PCA) for data exploration. In a scores plot, the treated and untreated biomass were distinguishable along the two main axes, PC1 and PC2. Examining the absorption bands corresponding to lignocellulosic components indicated that the acid pretreatment had resulted in dissolution and degradation of hemicelluloses and lignin, confirmed typically by disappearance of bands. The alkali treatment however was not as rigorous as the acid treatment, as some characteristic bands of hemicelluloses and lignin were enhanced, suggesting condensation of the degraded polysaccharides. The computer-assisted analysis of the FTIR spectra allowed efficient and simultaneous comparisons of lignocellulosic compositions present in various treated and untreated biomass. This represents an improvement relative to the conventional methods, since a large dataset can be handled efficiently and individual peaks can be examined.

Decentralized thermophilic biohydrogen: A more efficient and cost-effective process

Nonfood lignocellulosic biomass is an ideal substrate for biohydrogen production. By avoiding pretreatment steps (acid, alkali, or enzymatic), there is potential to make the process economical. Utilization of regional untreated lignocellulosic biomass by cellulolytic and fermentative thermophiles in a consolidated mode using a single reactor is one of the ways to achieve economical and sustainable biohydrogen production. Employing these potential microorganisms along with decentralized biohydrogen energy production will lead us towards regional and national independence having a positive influence on the bioenergy sector.

DECENTRALIZED THERMOPHILIC BIOHYDROGEN: A MORE EFFICIENT AND COST EFFECTIVE PROCESS

Nonfood lignocellulosic biomass is an ideal substrate for biohydrogen production. By avoiding pretreatment steps (acid, alkali, or enzymatic), there is potential to make the process economical. Utilization of regional untreated lignocellulosic biomass by cellulolytic and fermentative thermophiles in a consolidated mode using a single reactor is one of the ways to achieve economical and sustainable biohydrogen production. Employing these potential microorganisms along with decentralized biohydrogen energy production will lead us towards regional and national independence having a positive influence on the bioenergy sector.

Metabolic engineering of Thermobifida fusca for direct aerobic bioconversion of untreated lignocellulosic biomass to 1-propanol

Biofuel production from renewable resources can potentially address lots of social, economic and environmental issues but an efficient production method has yet to be established. Combinations of different starting materials, organisms and target fuels have been explored with the conversion of cellulose to higher alcohols (1-propanol, 1-butanol) being one potential target. In this study we demonstrate the direct conversion of untreated plant biomass to 1-propanol in aerobic growth conditions using an engineered strain of the actinobacterium, Thermobifida fusca. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase was added to T. fusca leading to 1-propanol production during growth on glucose, cellobiose, cellulose, switchgrass and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. These results represent the first demonstration of direct conversion of untreated lignocellulosic biomass to 1-propanol in an aerobic organism and illustrate the potential utility of T. fusca as an aerobic, cellulolytic bioprocess organism.

Simple Chemical Transformation of Lignocellulosic Biomass into Furans for Fuels and Chemicals

Lignocellulosic biomass is a plentiful and renewable resource for fuels and chemicals. Despite this potential, nearly all renewable fuels and chemicals are now produced from edible resources, such as starch, sugars, and oils; the challenges imposed by notoriously recalcitrant and heterogeneous lignocellulosic feedstocks have made their production from nonfood biomass inefficient and uneconomical. Here, we report that N,N-dimethylacetamide (DMA) containing lithium chloride (LiCl) is a privileged solvent that enables the synthesis of the renewable platform chemical 5-hydroxymethylfurfural (HMF) in a single step and unprecedented yield from untreated lignocellulosic biomass, as well as from purified cellulose, glucose, and fructose. The conversion of cellulose into HMF is unabated by the presence of other biomass components, such as lignin and protein. Mechanistic analyses reveal that loosely ion-paired halide ions in DMA-LiCl are critical for the remarkable rapidity (1-5 h) and yield (up to 92%) of this low-temperature (<or=140 degrees C) process. The simplicity of this chemical transformation of lignocellulose contrasts markedly with the complexity of extant bioprocesses and provides a new paradigm for the use of biomass as a raw material for a renewable energy and chemical industries.

Onopordum nervosum as biomass source: Some aspects of its production and transformation by enzymatic hydrolysis

Onopordum nervosum, a lignocellulosic herbaceous species of the Iberian Peninsula, has been selected as a suitable biomass source to be used in transformation processes to obtain energetic or industrial products.In this work, the effectiveness of different chemical pretreatments as a preliminary step to the enzymatic hydrolysis of this lignocellulosic biomass was evaluated. In order to determine biomass productivity, field assays were carried out in 1988 and 1989 using different planting densities and evaluating the effect of top fertilization. Biomass yields between 12 and 20 t ha −1 were obtained, depending on the year and the planting density assayed. No significant differences were found in production rates when top fertilization was applied. Enzymatic hydrolysis of O. nervosum using a cellulolytic complex from Trichoderma longibrachiatum QM9414, gave low yields when untreated lignocellulosic biomass was used as substrate. Among different chemical pretreatments tested, ethanol and butanol solubilizations in the presence of a basic catalyst gave the best results. For the most effective pretreatment conditions, a delignification of about 30% and a complete recovery of glucose in the treated substrate were obtained both for butanol and ethanol. The highest enzymatic hydrolysis yields were found when the ethanol was used as solvent, giving a saccharification efficiency of about 66% which, compared to the 23% for the native substrate, indicates the remarkable increment in the susceptibility of the cellulose to enzyme attack effected by this pretreatment.