Incorporation of canola meal as a sustainable natural filler in PLA foams.

The canola oil industry generates significant waste as canola meal (CM) which has limited scope and applications. This study demonstrates the possibility of valorization of CM as a sustainable natural filler in a biodegradable polymer composite of Poly(lactic acid) (PLA). Generally, interfacial bonding between natural fibers and the polymer matrix in the composite is weak and non-uniform. One possible solution is to derivatize natural fibre to introduce interfacial bond strength and compatibility with the PLA polymer matrix. Here, CM was succinylated in a reactive extrusion process using succinic anhydride at 30 wt% to get 14% derivatization with 0.02 g of -COOH density per g of CM. The CM or succinylated CM at 5 and 15 wt% was co-extruded with amorphous PLA to get composite fibers. CM-PLA and succinylated CM-PLA biocomposites were foamed using a mild and green microcellular foaming process, with CO2 as an impregnating agent without any addition of organic solvents. The properties of the foams were analyzed using differential scanning calorimetry (DSC), Dynamic mechanical thermal analysis (DMTA), shrinkage, and imaging. The addition of CM or succinylated CM as a natural filler did not significantly change the glass transition temperature, melting point, percent crystallization, stiffness, and thermal stability of PLA foams. This suggests succinylation (modification) of CM is not a mandatory step for improving interphase compatibility with the amorphous PLA. The new PLA-CM foams can be a good alternative in the packaging industry replacing the existing petroleum-based polymer foams.

Environmentally Benign Fast-Degrading Conductive Composites.

An environmentally benign conductive composite that rapidly degrades in the presence of warm water via enzyme-mediated hydrolysis is described. This represents the first time that hydrolytic enzymes have been immobilized onto eco-friendly conductive carbon sources with the express purpose of degrading the encapsulating biodegradable plastic. Amano Lipase (AL)-functionalized carbon nanofibers (CNF) were compounded with polycaprolactone (PCL) to produce the composite film CNFAL-PCL (thickness ∼ 600 µm; CNFAL = 20.0 wt %). To serve as controls, films of the same thickness were also produced, including CNF-AL5-PCL (CNF mixed with AL and PCL; CNF = 19.2 wt % and AL = 5.00 wt %), CNF-PCL (CNF = 19.2 wt %), ALx-PCL (AL = x = 1.00 or 5.00 wt %), and PCL. The electrical performance of the CNF-containing composites was measured, and conductivities of 14.0 ± 2, 22.0 ± 5, and 31.0 ± 6 S/m were observed for CNFAL-PCL, CNF-AL5-PCL, and CNF-PCL, respectively. CNFAL-PCL and control films were degraded in phosphate buffer (2.00 mg/mL film/buffer) at 50 °C, and their average percent weight loss (Wtavg%) was recorded over time. After 3 h CNFAL-PCL degraded to a Wtavg% of 90.0% and had completely degraded after 8 h. This was considerably faster than CNF-AL5-PCL, which achieved a total Wtavg% of 34.0% after 16 days, and CNF-PCL, which was with a Wtavg% of 7.00% after 16 days. Scanning electron microscopy experiments (SEM) found that CNFAL-PCL has more open pores on its surface and that it fractures faster during degradation experiments which exposes the interior enzyme to water. An electrode made from CNFAL-PCL was fabricated and attached to an AL5-PCL support to form a fast-degrading thermal sensor. The resistance was measured over five cycles where the temperature was varied between 15.0-50.0 °C. The sensor was then degraded fully in buffer at 50 °C over a 48 h period.

Woody biomass as a potential feedstock for fermentative gaseous biofuel production.

Biogas and biohydrogen are compatible gaseous biofuels that can be blended with natural gas for reticulated fuel supply to reduce greenhouse gas emissions. Sustainably grown woody biomass is emerging as a potential feedstock in the production of biofuels. Woody biomass is widely available, uses non-arable land for plantation, does not require synthetic fertilisers to grow and acts as a carbon sink. The cellulose and hemicellulose fractions of wood are renewable sources of sugars that can be used for fermentative production of gaseous biofuels. However, widespread use of wood as a gaseous biofuel feedstock is constrained due to the recalcitrant nature of wood to enzymatic hydrolysis. Pretreatment makes cellulose and hemicellulose accessible to microbial enzymes to produce fermentable sugars. Here we review wood composition, its structure and different pretreatment techniques in the context of their effects on deconstruction of wood to improve hydrolysis and fermentative gaseous fuel production. The anaerobic digestion of pretreated wood for biogas and dark fermentation for biohydrogen production are discussed with reference to gas yields. Key advancements in lab-scale research are described for pretreatments and for pure, co- and mixed culture fermentations. Limitations to yield improvements are identified and future perspectives and prospects of gaseous biofuel production from woody biomass are discussed, with reference to new developments in engineered biocatalysts and process integration.

3D-Printed Enzyme-Embedded Plastics.

A simple and environmentally friendly approach toward the thermoplastic processing of rapidly degradable plastic-enzyme composites using three-dimensional (3D) printing techniques is described. Polycaprolactone/Amano lipase (PCL/AL) composite films (10 mm × 10 mm; height [h] = ∼400 µm) with an AL loading of 0.1, 1.0, and 5.0% were prepared via 3D printing techniques that entail direct mixing in the solid state and thermal layer-by-layer extrusion. It was found that AL can tolerate in situ processing temperatures up to 130 °C in the solid-state for 60 min without loss of enzymatic activity. The composites were degraded in phosphate buffer (8 mg/mL, composite to buffer) for 7 days at 37 °C and the resulting average percent total weight loss (WLavg %) was found to be 5.2, 92.9, and 100%, for the 0.1, 1.0, and 5.0% films, respectively. The degradation rates of PCL/AL composites were found to be faster than AL applied externally in the buffer. Thicker PCL/AL 1.0% films (10 mm × 10 mm; h = ∼500 µm) were also degraded over a 7 day period to examine how the weight loss occurs over time with 3.0, 18.1, 36.4, 46.4, and 70.2% weight loss for days 1, 2, 3, 4, and 7, respectively. Differential scanning calorimetry (DSC) analysis shows that the film's percent crystallinity (Dxtal%) increases over time with Dxtal% = 46.5 for day 0 and 53.1% for day 7. Scanning electron microscopy (SEM) analysis found that film erosion begins at the surface and that water can penetrate the interior via surface pores activating the enzymes embedded in the film. Controlled release experiments utilizing dye-loaded PCL/AL/dye (AL = 1.0%; dye = 0.1%) composites were degraded over a 7 day period with the bulk of the dye released by the fourth day. The PCL/AL multimaterial objects containing AL-resistant polylactic acid (PLA) were also printed and degraded to demonstrate the application of this material on more complex structures.

Assessing the potential of purple phototrophic microbial community for nitrogen recycling from ammonia-rich medium and anaerobic digestate.

Purple phototrophic bacteria (PPB) community, enriched from municipal wastewater, was characterized to assess their growth, tolerance, composition and potential for resource recovery from NH4+-rich medium. Batch experiments were conducted in tissue culture flasks and glass bottles under anaerobic conditions with infra-red lights. PPBs showed remarkable tolerance to high concentrations of NH4+-N and acetate. Below 1.5 g/L, growth was unaffected by NH4+-N with optical density at 590 nm (OD590) reaching 2.6-2.9, while they could tolerate 4.5 g/L NH4+-N. Similarly, PPB growth was unaffected at acetate concentrations below 4 g/L and they could tolerate >20 g/L acetate. Taxonomic characterization showed that the community comprised of 37-52% PPBs (with 15-20% proteins) under different conditions, with Rhodobacter sp. over Rhodopseudomonas sp. dominating at higher NH4+-N concentrations. PPBs showed growth and removal rates in anaerobic digestate and accumulated 26% proteins. These results indicated the potential of PPBs in resource recovery from NH4+-rich wastewater.

Penicillium rotoruae, a new Species from an In-Ground Timber Durability Test Site in New Zealand.

A Penicillium species isolated during a 1960s study on the ecology of fungi infecting Pinus radiata timber, and subsequently held in an in-house collection in Rotorua, New Zealand, was found to differ morphologically and in growth rate from two closely related Penicillium species. Phylogenetic analysis of the rDNA internal transcribed spacer (ITS), ß-tubulin, calmodulin and RNA polymerase II second largest subunit regions (RPB2) confirmed this to be a new species closely related to Penicillium ochrochloron in the Rolfsiorum series of the Lanata-Divaricata section and Aspergilloides sub-genus. Micromorphologically, the new species is characterised by predominantly monoverticilliate and occasional divaricate or biverticilliate conidiophores and smooth-walled subglobose to slightly ovoid conidia with absence of conidiogenesis at 25 °C. This new species is described here as Penicillium rotoruae sp. nov. which has potential applications in biofuel and biorefining industry.

Synthesis of graft copolymers of chitosan-poly(caprolactone) by lipase catalysed reactive extrusion.

Chitin is an abundant natural polymer and its deacetylated derivative chitosan has been a focus for the development of biobased, biocompatible and antimicrobial materials. In this work, a green and scalable route to grafting polycaprolactone (PCL) to chitosan using an enzyme catalysed reactive extrusion process is described. FTIR, 1H and 13C NMR spectroscopy and HSQC analysis confirm grafting of PCL to chitosan and show differences in the grafting pattern obtained using two commercially produced lipase enzymes from Candida antarctica (CALB® and NovoCor®). The thermostable NovoCor enzyme gave a much higher grafting yield (96.3%) than the less thermostable CALB enzyme (5.90%). In the esterification reaction, CALB preferentially catalyses reaction on primary OH groups at the C-6 position of chitosan, whereas NovoCor catalyses on the secondary OH groups of chitosan at the C-3 position. This is related to the differences in the selectivity of the two lipase enzymes. The control synthesized without enzyme did not show any grafting reaction. The degree of crystallinity and thermal stability of the lipase catalysed copolymer was reduced compared to unmodified chitosan. Moreover, the PCL grafted chitosan produced by a solvent free reactive extrusion route retained antimicrobial property against E.coli. Such grafted co-polymers may have applications in the controlled release coatings and tissue culture surfaces.

Versatile catechol dioxygenases in Sphingobium scionense WP01T.

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413-416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.

A mild thermomechanical process for the enzymatic conversion of radiata pine into fermentable sugars and lignin.

BACKGROUND: Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route. RESULTS: We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine (Pinus radiata) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3-72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained ß-ether interunit linkages, providing an opportunity for conversion into saleable co-products. CONCLUSIONS: This process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up.

Visualising recalcitrance by colocalisation of cellulase, lignin and cellulose in pretreated pine biomass using fluorescence microscopy.

Mapping the location of bound cellulase enzymes provides information on the micro-scale distribution of amenable and recalcitrant sites in pretreated woody biomass for biofuel applications. The interaction of a fluorescently labelled cellulase enzyme cocktail with steam-exploded pine (SEW) was quantified using confocal microscopy. The spatial distribution of Dylight labelled cellulase was quantified relative to lignin (autofluorescence) and cellulose (Congo red staining) by measuring their colocalisation using Pearson correlations. Correlations were greater in cellulose-rich secondary cell walls compared to lignin-rich middle lamella but with significant variations among individual biomass particles. The distribution of cellulose in the pretreated biomass accounted for 30% of the variation in the distribution of enzyme after correcting for the correlation between lignin and cellulose. For the first time, colocalisation analysis was able to quantify the spatial distribution of amenable and recalcitrant sites in relation to the histochemistry of cellulose and lignin. This study will contribute to understanding the role of pretreatment in enzymatic hydrolysis of recalcitrant softwood biomass.

Micromorphological changes and mechanism associated with wet ball milling of Pinus radiata substrate and consequences for saccharification at low enzyme loading.

In this work, substrates prepared from thermo-mechanical treatment of Pinus radiata chips were vibratory ball milled for different times. In subsequent enzymatic hydrolysis, percent glucan conversion passed through a maximum value at a milling time of around 120min and then declined. Scanning electron microscopy revealed breakage of fibers to porous fragments in which lamellae and fibrils were exposed during ball milling. Over-milling caused compression of the porous fragments to compact globular particles with a granular texture, decreasing accessibility to enzymes. Carbon-13 NMR spectroscopy showed partial loss of interior cellulose in crystallites, leveling off once fiber breakage was complete. A mathematical model based on observed micromorphological changes supports ball milling mechanism. At a low enzyme loading of 2FPU/g of substrate and milling time of 120min gave a total monomeric sugar yield of 306g/kg of pulp which is higher than conventional pretreatment method such as steam exploded wood.

Green route to modification of wood waste, cellulose and hemicellulose using reactive extrusion.

A large volume of wood waste is produced in timber processing industry which traditionally used in low value applications. Here, value addition to the wood waste (Sander dust) and cellulose, hemicellulose isolated thereof by functionalisation using cyclic anhydrides in a solvent-free and green reactive extrusion process is reported. The effect of extrusion temperature, catalyst and different weight ratios of Sander dust (SD):succinic anhydride (SA) on the esterification reaction is evaluated. The esterified products were characterised by the acid value, degree of substitution (DS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), solid state (13)C NMR and thermo-gravimetric analysis (TGA). Under optimum extrusion conditions, mixed esters are formed, with highest acid value obtained for succinylation of cellulose (0.122 g/g at DS of 0.350) which is two times higher compared to succinylated SD (0.059 g/g at a weight gain of 0.452) and hemicellulose (0.043 g/g at DS of 0.290). The reactivity trend for individual anhydride was: (1) SA-Cellulose>SD>hemicellulose; (2) maleic anhydride (MA)-SD>hemicellulose>cellulose and (3) dodecenyl succinic anhydride (DDSA)-SD ≈ cellulose ≫ hemicellulose. The pendant free carboxyl groups generated through functionalisation of wood waste, cellulose and hemicellulose without the presence of polymeric carriers will allow more tailored or targeted modification of wood-plastic composites.

Improved saccharification of steam-exploded Pinus radiata on supplementing crude extract of Penicillium sp.

Commercially available enzymes do not contain all the necessary softwood-specific accessory enzymes to obtain high saccharification efficiency. In this work, six saprophytic fungi obtained from Pinus radiata plantation site were screened for the putative softwood-specific accessory enzyme, ß-mannanase. A Penicillium sp. was found to produce ß-mannanase in both solid (31.6 units/g of dry biomass) and liquid (117 units/g of dry biomass) cultures using locust bean gum as an inducer after 2 weeks of incubation. The saccharification of steam-exploded Pinus radiata was 7.8 % w/w improved when the crude extract of Penicillium sp. was added to a mixture of commercial enzymes.

Nanoscale interactions of polyethylene glycol with thermo-mechanically pre-treated Pinus radiata biofuel substrate.

Non-productive adsorption of cellulose degrading enzymes on lignin is a likely reason for reduced rate and extent of enzymatic conversion of lignocellulosic substrate to sugars. Additives such as polyethyleneglycol (PEG) may act as blocking agents in this non-productive interaction. However, the exact molecular level interactions of PEG with lignin in pre-treated lignocellulosic substrates are not known. We have used confocal fluorescence microscopy combined with Förster resonance energy transfer (FRET) to reveal molecular level interactions between lignin present in thermo-mechanically pre-treated Pinus radiata substrate, and fluorescently labeled PEG. It is demonstrated that PEG interaction with lignin is mainly associated with particles derived from secondary walls, with little or no penetration into fragments derived from the middle lamella. This nanoscale information on the PEG-substrate interaction will assist in rationalizing pre-treatment methods to reduce the recalcitrance of softwood biofuel substrates.

A mathematical model for the inhibitory effects of lignin in enzymatic hydrolysis of lignocellulosics.

A new model for enzymatic hydrolysis of lignocellulosic biomass distinguishes causal influences from enzyme deactivation and restrictions on the accessibility of cellulose. It focuses on calculating the amount of unreacted cellulose at cessation of enzyme activity, unlike existing models that were constructed for calculating the time dependence of conversion. There are three adjustable parameters: (1) 'occluded cellulose' is defined as cellulose that cannot be hydrolysed regardless of enzyme loading or incubation time, (2) a 'characteristic enzyme loading' is sufficient to hydrolyse half of the non-occluded cellulose, (3) a 'mechanism index' measures deviations from first-order kinetics. This model was used to predict that the optimal incubation temperature is lower for lignocellulosics than for pure cellulose. For steam-exploded pine wood after 96h incubation, occluded cellulose was 24% and 26% at 30°C and 50°C, and the characteristic enzyme loadings were 10 and 18FPU/g substrate, respectively.

Optimizing the enzyme loading and incubation time in enzymatic hydrolysis of lignocellulosic substrates.

A mathematical model for costing enzymatic hydrolysis of lignocellulosics is presented. This model is based on three variable parameters describing substrate characteristics and three unit costs for substrate, enzymes and incubation. The model is used to minimize the cost of fermentable sugars, as intermediate products on the route to ethanol or other biorefinery products, by calculating optimized values of enzyme loading and incubation time. This approach allows comparisons between substrates, with processing conditions optimized independently for each substrate. Steam-exploded pine wood was hydrolyzed in order to test the theoretical relationship between sugar yield and processing conditions.

Correlative light and scanning electron microscopy of the same sections gives new insights into the effects of pectin lyase on bordered pit membranes in Pinus radiata wood.

Bordered pits are structures in the cell walls of softwood tracheids which permit the movement of water between adjacent cells. These structures contain a central pit membrane composed of an outer porous ring (margo) and an inner dense and pectin-rich disc (torus). The membrane is overarched on each side by pit borders. Pits may be aspirated, a condition where the torus seals against the pit border, effectively blocking the pathway between cells. In living trees this maintains overall continuity of water conduction in xylem by sealing off tracheids containing air. Drying of timber results in further pit aspiration, which reduces wood permeability to liquid treatment agents such as antifungal chemicals. One possible way to increase permeability is by treating wood with pectin lyase to modify or remove the torus. The effectiveness of this treatment was initially evaluated using light microscopy (LM) of toluidine blue stained wood. Pectic material is coloured pink-magenta with this stain, and loss of this colour after treatment has been interpreted as indicating destruction of the torus. However, correlative light (LM) and scanning electron (SEM) microscopic observations of identical areas of toluidine blue stained sections revealed that many unstained pits had intact but modified tori when viewed with SEM. These observations indicate that LM alone is not sufficient to evaluate the effects of pectin lyase on pit membranes in wood. Combining LM and SEM gives more complete information.

Pre-treatment of Pinus radiata substrates by basidiomycetes fungi to enhance enzymatic hydrolysis.

Pre-treatment is important step prior to enzymatic hydrolysis of ligno-cellulosic biomass in order to obtain renewable carbon source ca. glucose. Pinus radiata biomass including wood blocks, wood chips and steam exploded wood (SEW) were used to investigate the effect of fungal pre-treatment on glucose yield. Comparison was made using one white-rot fungus (Trametes versicolor) and three brown-rot fungi (Coniophora puteana, Antrodia xantha and Oligoporus placenta). This is the first study where SEW was treated with basidiomycetes and subsequent enzymatic hydrolysis gave 5 g glucose/l which is an order of magnitude greater compared to control biomass (0.5 g glucose/l). This enhanced glucose yield is due to the novel pre-treatment sequence used in this study.