Two thermostable hemicellulases and their effects on wood pulps

Two hemicellulases, cloned from a thermophilic anaerobe (isolate TP8 6.3.3.1) were partially purified and characterised. The ability of these enzymes to hydrolyse the hemicellulose components of various types of pulps and their effects on the bleachability of radiata pine kraft pulp were examined. The xylanase (two preparations) and mannanase had broad pH optima (6-7.5). The xylanases had temperature optima of 70°C (10 min assay) and the mannanase had an optimum of 80°C (10 min assay). All the cloned enzymes were thermostable with more than 95% of the enzyme activity remaining after 24 hours at 70°C, in an unbuffered solution. Buffering affected the thermostability of the enzymes. The mannanase had a half life of 48 min at 85°C. The xylanases were able to hydrolyse xylans but also had a pH-dependent CM cellulase activity, but this was never more than 10% of the xylanase activity. The mannanase hydrolysed mannan, glucomannans and galactomannans. The glucose content of the glucomannans had little or no effect on mannanase activity but the galactose substituents of the galactomannans severely inhibited the hydrolysis of these substrates. The thermostable xylanase hydrolysed more than 75% of soluble radiata pine xylan to low molecular weight (< DP10) xylo-oligomers. These included; xylose, xylobiose, xylotriose, arabinose-substituted xylobiose and xylotriose and 4-O-methyl glucuronic acid substituted xylotrioses. The arabinose substituents were bound to the terminal, non-reducing xylose residue; the glucuronic acid substituents may be bound to the non-reducing or central xylose residues of the substituted xylotriose. The thermostable mannanase also hydrolysed more than 75% of soluble radiata glucomannan, producing mannose, mannobiose, mannotriose, glucomannose and glucomannobiose. The hemicellulases were used to treat (at 70°C, 24 hr) a variety of wood pulps, including kraft (softwood and hardwood), radiata NSAQ pulp, radiata CTMP and radiata TMP. Both enzymes solubilised their respective substrates from these pulps. The chemical pulps were more susceptible to enzymatic attack than the mechanical pulps, possibly due to the greater fibre porosity of the chemical pulps. The xylanase solubilised ~4% of the xylan and the mannanase solubilised 10% of the glucomannan from radiata kraft pulp. In comparison with other xylanases, the thermostable xylanase was less effective in solubilising xylan from kraft pulps. The hemicelluloses of kraft pulps that had been enzyme treated were extracted. The extracted xylan from the xylanase-treated pulp was lower in average DP than the xylan extracted from untreated pulps. The average DP of the extracted glucomannan from the mannanase-treated pulps was no different than that of glucomannans extracted from untreated pulps. The extracted xylan and glucomannan from untreated kraft pulps were readily hydrolysed by the xylanase and the mannanase respectively, suggesting that enzyme inaccessibility to the substrate within the fibre wall was the main cause of their resistance to enzymatic attack. The effect of these enzymes on the bleachability of radiata kraft pulps was studied by measuring the reduction in kappa number (κ), relative to untreated controls, after a standard D/C E prebleaching sequence. The xylanase and the mannanase had little or no effect on the bleachability of radiata kraft pulps. Xylanase treated kraft pulp required a protease treatment after the hemicellulose treatment but before bleaching, in order to elicit any improvement in bleachability (~7% drop in κ relative to untreated control pulps). Mannanase treated kraft pulp showed no improvement in bleachability. For comparison commercial xylanases (including a thermostable xylanase) were used to treat radiata kraft pulps and did improve bleachability significantly. These enzymes also solubilised three to four times more xylan than the cloned, thermostable xylanase. Therefore, the ineffectiveness of the cloned xylanase may be due to its very limited ability to hydrolyse intra-fibril xylan in kraft pulp fibres

Predicting Wood Decay and Density Using Nir Spectroscopy

Variable wood chip density and extent of decay can significantly affect pulp yield and quality. Rapid methods are needed to quantify and eventually control these variables. Near infrared (NIR) spectroscopy is a rapid technique that may be suitable for measuring these variables. In the present research, NIR spectroscopy was used to develop partial least squares (PLS) models capable of estimating basic wood density and extent of decay in wood. These methods offer a rapid means of determining wood density and extent of decay, and can be applied to routine lab analyses as well as online measurements for process control applications

Kraft Pulping of Softwood Chips with Mild Hot Water Pre-hydrolysis to Understand the Effects of Wood Chip Thickness

Hemicelluloses consume alkali during kraft pulping and dissolve in the black liquor as a low energy fuel. Acidic pre-hydrolysis of softwood chips removes hemicelluloses but preserves cellulose content prior to pulping. This study compared mild pre-hydrolysis (140 °C) kraft pulping with conventional kraft pulping of commercial softwood chips at two H-factors for wood chips with thickness ranging from less than 2 mm to over 6 mm. The chip thickness less than 2 mm increased hemicelluloses oligomer yield and showed little influence on pulp fiber yield. However, the kraft pulp fiber length decreased 5.6%. Kappa number and fiber reject increased dramatically when chip thickness was greater than 6 mm. The detailed compositional analysis of kraft pulp and fiber quality analysis indicate that pre-hydrolysis followed by kraft pulping enhanced delignification with limited reduction of fiber length and width, and increased kinks. Strategic considerations for the integration of pre-hydrolysis into kraft pulping for future biorefineries were outlined

The characterisation of a thermostable endo-β-1,4-mannanase cloned from “Caldocellum saccharolyticum”

An endo-gb-1,4-mannanase cloned from caldocellum saccharolyticum and expressed in Escherichia coli was partially purified. The purification involved heat treatment, anion exchange and gel filtration. The mannanase was only active against mannan, glucomannans and galactoglucomannans and obeyed Michaelis-Menten kinetics on these substrates. The rate and extent of hydrolysis was dependent on the type of substrate. Galactomannans were not as readily depolymerized as the mannan and glucomannans investigated. The glucose content of the glucomannans did not affect the rate of hydrolysis and only slightly affected the extent. The molecular mass of the mannanase was estimated at 39 kDa. The pH and temperature optima were 6.5 and 80° C respectively. The mannanase was very thermostable with a half life of 48 min at 85° C and no loss in activity after 24 h at 70° C

Mesolithic human occupation and seasonality: sclerochronology, δ 18 O isotope geochemistry, and diagenesis verification by Raman and LA-ICP-MS analysis of Argyrosomus regius (meagre) sagittae otoliths from layer 1 of Cabeço da Amoreira Mesolithic shell midden (Muge, Portugal)

We present preliminary sclerochronological analysis on 15 Argyrosomus regius (meagre) otoliths collected from two different human occupation levels from the Cabeço da Amoreira shell midden (Muge valley, Portugal). The otoliths were sectioned and observed under a reflected light stereomicroscope to examine seasonal growth rings. Carbonates collected from individual growth rings subsampled with a micromill sampling device were analyzed for their stable oxygen isotope content with the objective of determining the predominant season of capture and therefore season of site use, environmental conditions, and sites of resource procurement (local versus regional). The otoliths’ stable isotope records show clear seasonality and a season of capture estimate that seems consistent with a “good season” (warmer season, i.e., from spring to late summer/beginning of autumn in this area) site use in the last occupation layer (the great majority of otoliths are from layer 1), except for four samples giving heaver oxygen isotope (δ18O) values that suggest colder conditions and only one with markedly positive δ18O values. Potential effect of diagenesis on the otolith records was also assessed through coupled laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) and by micro-X-Ray diffraction (μ-XRD). Results revealed the presence of only aragonite and no traces of calcite, providing no evidence of diagenesis that could significantly alter isotope results and lead to erroneous interpretations. The implications of these results are discussed and compared with data from other archeological sites, as well as data from micromorphology regarding continuity or interruption of site use and other faunal remains

Association Genetics of Wood Physical Traits in the Conifer White Spruce and Relationships With Gene Expression

Marker-assisted selection holds promise for highly influencing tree breeding, especially for wood traits, by considerably reducing breeding cycles and increasing selection accuracy. In this study, we used a candidate gene approach to test for associations between 944 single-nucleotide polymorphism markers from 549 candidate genes and 25 wood quality traits in white spruce. A mixed-linear model approach, including a weak but nonsignificant population structure, was implemented for each marker–trait combination. Relatedness among individuals was controlled using a kinship matrix estimated either from the known half-sib structure or from the markers. Both additive and dominance effect models were tested. Between 8 and 21 single-nucleotide polymorphisms (SNPs) were found to be significantly associated (P ≤ 0.01) with each of earlywood, latewood, or total wood traits. After controlling for multiple testing (Q ≤ 0.10), 13 SNPs were still significant across as many genes belonging to different families, each accounting for between 3 and 5% of the phenotypic variance in 10 wood characters. Transcript accumulation was determined for genes containing SNPs associated with these traits. Significantly different transcript levels (P ≤ 0.05) were found among the SNP genotypes of a 1-aminocyclopropane-1-carboxylate oxidase, a β-tonoplast intrinsic protein, and a long-chain acyl-CoA synthetase 9. These results should contribute toward the development of efficient marker-assisted selection in an economically important tree species

Validation of a stereo camera system to quantify brain deformation due to breathing and pulsatility

PURPOSE: A new stereo vision system is presented to quantify brain shift and pulsatility in open-skull neurosurgeries. METHODS: The system is endowed with hardware and software synchronous image acquisition with timestamp embedding in the captured images, a brain surface oriented feature detection, and a tracking subroutine robust to occlusions and outliers. A validation experiment for the stereo vision system was conducted against a gold-standard optical tracking system, Optotrak CERTUS. A static and dynamic analysis of the stereo camera tracking error was performed tracking a customized object in different positions, orientations, linear, and angular speeds. RESULTS: The system is able to detect an immobile object position and orientation with a maximum error of 0.5 mm and 1.6° in all depth of field, and tracking a moving object until 3 mm/s with a median error of 0.5 mm. Three stereo video acquisitions were recorded from a patient, immediately after the craniotomy. The cortical pulsatile motion was captured and is represented in the time and frequency domain. The amplitude of motion of the cloud of features' center of mass was inferior to 0.8 mm. Three distinct peaks are identified in the fast Fourier transform analysis related to the sympathovagal balance, breathing, and blood pressure with 0.03-0.05, 0.2, and 1 Hz, respectively. CONCLUSIONS: The stereo vision system presented is a precise and robust system to measure brain shift and pulsatility with an accuracy superior to other reported systems