Cellulose-rich Substrates Research Articles

Heavy water-labeled Raman spectroscopy reveals carboxymethylcellulose-degrading bacteria and degradation activity at the single-cell level.

Biodegradation of cellulose-rich substrates is an indispensable process for soil carbon replenishment in various ecological niches. Biodegradation of cellulose has been studied extensively via an enzyme assay to quantify the amount of cellulase with a view to identify effective cellulose degraders. However, a bulk enzyme assay undermines the role of physiological heterogeneity between cells; it is therefore imperative to opt out for a more effective method such as single-cell Raman spectroscopy combined with heavy water (D2O) to reveal active cellulose degraders. Cellular incorporation of D2O-derived D produces a new C-D Raman band which can act as a quantitative indicator of microbial activity. In this study, metabolic responses of seven cellulose-degrading bacteria to carboxymethylcellulose (CMC) and glucose were evaluated via the C-D Raman band. On the basis of % C-D, CMC was demonstrated to be most efficiently metabolized by Bacillus velezensis 2a-9 and Providencia vermicola 5a-9(b). Metabolic activity between individual cells of B. velezensis and P. vermicola towards CMC ranged from approximately 8 to 27% and 6 to 16%, respectively, clearly indicating heterogeneous degradation activities among isogenic populations. Linear correlation between % C-D and specific endoglucanase activity validated Raman results on CMC-degrading activity. Also, % C-D obtained from bacteria cultivated with only glucose was around 60% higher than that obtained from CMC, indicating the preference of bacteria for simple sugar glucose than CMC. In conclusion, Raman spectroscopy combined with heavy water is a sensitive analytical technique to reveal cellulose degraders and their degrading activities.

Comparative study of hemicelluloses from Hybrid Pennisetum via a green and clean integrated process

In this study, a green and clean integrated process based on microwave-assisted hydrothermal pretreatment (MHTP) and subsequent alkali post-treatment was performed to isolate and characterize water- and alkali-soluble hemicelluloses (SHs and AHs) from Hybrid Pennisetum (HP) as well as to improve the enzymatic digestibility of HP. Meanwhile, part of hemicelluloses was degraded into the value-added xylo-oligosaccharides (XOS) during the MHTP process, which would be applied to the food and pharmaceuticals industries. State-of-the-art analytical techniques (e.g., HPLC, HPAEC, GPC, and 2D-HSQC) were used to investigate the main component streams (SHs, AHs, XOS, and glucose). Results showed that the structural characteristic of SHs and AHs was mainly composed of β-(1→4)-linked xylans. Additionally, the enzymatic digestibility of the cellulose-rich substrate (A190(10)) was 92.8%, which was 3.4 times higher than that of raw material (27.4%). In short, the proposed integrated process was considered as a promising biorefinery strategy for both hemicellulose-based products and bioethanol.

A comparison of various lignin-extraction methods to enhance the accessibility and ease of enzymatic hydrolysis of the cellulosic component of steam-pretreated poplar

BackgroundCurrent single-stage delignification-pretreatment technologies to overcome lignocellulosic biomass recalcitrance are usually achieved at the expense of compromising the recovery of the polysaccharide components, particularly the hemicellulose fraction. One way to enhance overall sugar recovery is to tailor an efficient two-stage pretreatment that can pre-extract the more labile hemicellulose component before subjecting the cellulose-rich residual material to a second-stage delignification process. Previous work had shown that a mild steam pretreatment could recover >65% of the hemicellulose from poplar while limiting the acid-catalysed condensation of lignin. This potentially allowed for subsequent lignin extraction using various lignin solvents to produce a more accessible cellulosic substrate.ResultsA two-stage approach using steam and/or solvent pretreatment was assessed for its ability to separate hemicellulose and lignin from poplar wood chips while providing a cellulose-rich fraction that could be readily hydrolysed by cellulase enzymes. An initial steam-pretreatment stage was performed over a range of temperatures (160–200 °C) using an equivalent severity factor of 3.6. A higher steam temperature of 190 °C applied over a shorter residence time of 10 min effectively solubilized and recovered 75% of the hemicellulose while enhancing the ability of various solvents [deep eutectic solvent (DES), ethanol organosolv, soda/anthraquinone (soda/AQ) or a hydrotrope] to extract lignin in a second stage. When the second-stage treatments were compared, the mild DES treatment (lactic acid and betaine) at 130 °C, removed comparable amounts of lignin with higher selectivity than did the soda/AQ and organosolv pretreatments at 170 °C. However, the cellulose-rich substrates obtained after the second-stage organosolv and soda/AQ pretreatments showed the highest cellulose accessibility, as measured by the Simon’s staining technique. They were also the most susceptible to subsequent enzymatic hydrolysis.ConclusionsThe second-stage pretreatments varied in their ability to solubilize and extract the lignin component of steam-pretreated poplar while enhancing the enzymatic hydrolysis of the resulting cellulose-rich residual fractions. Although DES extraction was more selective in extracting lignin from the steam-pretreated substrates, the organosolv and soda/AQ post treatments disrupted the cellulose structure to a greater extent while enhancing the ease of enzymatic hydrolysis. Graphical abstractEffective hemicellulose removal via steam pretreatment followed by subsequent lignin extraction under acidic, alkaline or solvolytic conditions results in a highly accessible, more readily hydrolysed cellulose fraction.

Hydrolytic potential of five fungal supernatants to enhance a commercial enzyme cocktail

ObjectivesTo evaluate the potential of enzyme cocktails produced by five filamentous fungi to supplement the industrial cellulase cocktail, Celluclast 1.5L, in order to improve the efficiency of saccharification.ResultsThe fungi were cultivated on wheat bran and the resulting supernatants were combined with Celluclast in enzymatic hydrolysis experiments to test their ability to hydrolyze wheat bran and five cellulose-rich substrates. The supernatant showing the best performance was that from an Aspergillus niger cellulase mutant. The addition of β-glucosidase only to the Celluclast cocktail was not as beneficial.ConclusionSupplementing commercial cocktails with enzymes from carefully selected fungi may result in significantly more efficient saccharification of lignocellulosic materials. Furthermore, such an approach could lead to the identification of novel enzyme activities crucial for saccharification.

Feedstock selection for polymer and chemical production: feedstock-specific recalcitrance.

Plant cell wall materials derived from a range of waste biomass sources have great potential as a source of sustainable alternatives to petrochemicals. Perhaps the most straightforward way of realising this potential would be to hydrolyse the most efficiently fermentable polymers into their constituent sugars and use yeast to ferment these into useful chemicals. However, it also makes sense to pre-extract components which have a greater value in polymeric form. This is particularly true for non-cellulosic polymers, which are rich in poorly-fermentable pentose sugars. Liquid hot water (LHW) pretreatment can be used to extract non-cellulosic carbohydrates in a cost-effective manner, leaving a cellulose-rich substrate which is easier to hydrolyse using commercial cellulases. However, inherent differences in the plant cell wall structure and composition mean that some biomass sources may be more suitable for exploitation than others. Here, we examine eight different feedstocks (two each from hardwood, softwood, cereal straws and dicotyledonous crops), expose them to 26 different LHW pretreatment conditions and hydrolyse the entire pretreated slurry with a commercial cellulase. This enables side-by-side comparisons, in terms of saccharification yield, of the feedstocks. The results clearly demonstrate considerable differences in suitability between the feedstocks, in relation to the quantity of products released and the processes needed to obtain them.

Synergetic effect of dilute acid and alkali treatments on fractional application of rice straw.

BackgroundThe biorefinery based on an effective and economical process is to fractionate the three primary constituents (cellulose, hemicelluloses, and lignin) from lignocellulosic biomass, in which the constituents can be respectively converted into high-value-added products. In this study, a successive treatment with dilute acid (0.25–1.0 % aqueous H2SO4, 100–150 °C, 0.5–3.0 h) and alkali (1.5 % aqueous NaOH, 80 °C, 3 h) was performed to produce xylooligosaccharides (XOS), high-purity lignin, and cellulose-rich substrates to produce glucose for ethanol production from rice straw (RS).ResultsDuring the dilute acid pretreatment, the maximum production of XOS (12.8 g XOS/100 g RS) with a relatively low level of byproducts was achieved at a relatively low temperature (130 °C) and a low H2SO4 concentration (0.5 %) for a reaction time of 2.0 h. During the alkali post-treatment, 14.2 g lignin with a higher purity of 99.2 % and 30.3 g glucose with a higher conversion rate by enzymatic hydrolysis were obtained from the successively treated substrates with 100 g RS as starting material. As the pretreatment temperature, H2SO4 concentration, or time increased, more β-O-4 linkages in lignins were cleaved, which resulted in an increase of phenolic OH groups in lignin macromolecules. The signal intensities of G2 and G6 in HSQC spectra gradually reduced and vanished, indicating that a condensation reaction probably occurred at C-2 and C-6 of guaiacyl with the side chains of other lignin.ConclusionsThe present study demonstrated that the successive treatments with dilute acid and alkali had a synergetic effect on the fractionation of the three main constituents in RS. It is believed that the results obtained will enhance the availability of the combined techniques in the lignocellulosic biorefinery for the application of the main components, cellulose, hemicelluloses, and lignin as biochemical and biofuels.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0632-9) contains supplementary material, which is available to authorized users.

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

Enzymatic hydrolysis of cellulose provides a renewable source of monosaccharides for production of variety of biochemicals and biopolymers. Unfortunately, the enzymatic hydrolysis of cellulose is often incomplete, and the reasons are not fully understood. We have monitored enzymatic hydrolysis in terms of molecular density, ordering and autofluorescence of cellulose structures in real time using simultaneous CARS, SHG and MPEF microscopy with the aim of contributing to the understanding and optimization of the enzymatic hydrolysis of cellulose. Three cellulose-rich substrates with different supramolecular structures, pulp fibre, acid-treated pulp fibre and Avicel, were studied at microscopic level. The microscopy studies revealed that before enzymatic hydrolysis Avicel had the greatest carbon-hydrogen density, while pulp fibre and acid-treated fibre had similar density. Monitoring of the substrates during enzymatic hydrolysis revealed the double exponential SHG decay for pulp fibre and acid-treated fibre indicating two phases of the process. Acid-treated fibre was hydrolysed most rapidly and the hydrolysis of pulp fibre was spatially non-uniform leading to fractioning of the particles, while the hydrolysis of Avicel was more than an order of magnitude slower than that of both fibres.

Structural changes in lignin during integrated process of steam explosion followed by alkaline hydrogen peroxide of Eucommia ulmoides Oliver and its effect on enzymatic hydrolysis

Eucommia ulmoides Oliver (EU) wood was successively treated by a combined system based on steam explosion pretreatment (SEP) and alkaline hydrogen peroxide post-treatment (AHPP). In this case, SEP was to disrupt the lignocellulosic structure, and the subsequent AHPP process was to isolate the high-purity lignin and cellulose-rich substrates. Results showed that the lignin fractions obtained during the AHPP exhibited smaller molecular weights, narrow polydispersity, less phenolic OH groups and lower syringyl/guaiacyl ratios (S/G) than those of the milled wood lignin (SEMWL) obtained from the only steam exploded EU. NMR characterization of lignin revealed that the AHPP process has a slight effect on the composition and molecular characteristic of lignin, and the lignin isolated had lower amounts of substructures (aryl-β-ether, resinol, and phenylcoumaran linkages) as compared to those in SEMWL. Moreover, the subsequent SEP followed by AHPP process enhanced the enzymatic hydrolysis of cellulose-rich substrates to a maximum value of 91.69%. It was found that the synergistic treatment removed most of lignin, degraded hemicelluloses, and incurred a higher crystalline index and surface area of the cellulose-rich substrates as compared to the only steam explosion pretreatment. The combination of the SEP and AHPP processes is an environmentally benign and advantageous scheme for the production of high-purity lignin and cellulose-rich substrates, which will be further transformed into the value-added biomaterials and bioethanol.

Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates

The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel® and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS 13C-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.

Anaerobic solid-phase fermentation of plant substrates by Bacillus subtilis

Solid-phase growth of Bacillus subtilis 8130 on cellulose-rich plant substrates (presscakes or pulp) under hypoxic conditions was accompanied by cellulose depolymerization, protein hydrolysis, and degradation of other plant components, including some processes of mixed-type carbohydrate fermentation. The bacterial fermentation yielded propionic, butyric, and hexanoic acids and butyric acid derivatives. The bacterial metabolism and fermentation degree can be characterized by the proportions of fatty acids in the reaction mixture. The product of sea buckthorn cake fermentation has a good sorption quality.

Xylanase production by a newly isolated Aspergillus foetidus strain and its characterization

Xylanase production by a newly isolated Aspergillus foetidus MTCC 4898 was studied using submerged fermentation under shaking. The maximum xylanase yield (210 U/ml) was obtained with negligible cellulase activity at 30 °C using 1% birchwood xylan as substrate in 3 days of cultivation. The strain was able to produce extracellular xylanases on birchwood xylan as well as on cellulose rich substrates also with very poor cellulase production (<0.1 FPU/ml). The partially purified enzyme recovered by ammonium sulphate fractionation showed maximum activity at 50 °C and at pH 5.3. The enzyme was highly stable at 40 °C but retained 36% of its activity at 50 °C after 3 h. The presence of KCl, CaCl 2, glycerol, xylan and trehalose significantly improved thermostability at 50 and 60 °C. Zymogram analysis indicated three isoenzymes. The enzyme preparation was completely stable at freezing temperature for up to 6 months and reasonably stable at refrigeration temperatures for more than 2 weeks.

Formation of xylanase by Schizophyllum commune: Effect of medium components

Growth of a wild strain of Schizophyllum commune on cellulose or cellulose-rich substrates in submerged culture resulted in the production of cellulase, xylanase, and mannanase activities. Pure xylan or galactomannan as carbon sources did not specifically induce the formation of xylanase or mannanase, respectively. Production of cellulase, xylanase, and mannanase activities decreased with an increasing ratio of xylan to cellulose, or mannan to cellulose, in the growth medium. During growth experiments, formation of xylanase, cellulase, as well as mannanase was induced not only by cellulose, but also by cellobiose, lactose, and l-sorbose. Xylans from birchwood, xylose, and β-methyl- d-xyloside, a structural analogue of xylobiose, did not induce xylanase formation when employed as substrates. These results indicate that the synthesis of xylanase, cellulase, and mannanase activities is likely to be under common regulatory control in the case of Schizophyllum commune. The inducer seems to be a small molecule derived from cellulose. Constitutive levels of xylanase and cellulase activities were detected during growth of the fungus on easily metabolizable substrates such as glucose, xylose, or glycerol. Among various organic nitrogen sources tested, yeast extract was found to be optimal for the production of xylanase.