Suitability of magnetic nanoparticle immobilised cellulases in enhancing enzymatic saccharification of pretreated hemp biomass

Previous research focused on pretreatment of biomass, production of fermentable sugars and their consumption to produce ethanol. The main goal of the work was to economise the production process cost of fermentable sugars. Therefore, the objective of the present work was to investigate enzyme hydrolysis of microcrystalline cellulose and hemp hurds (natural cellulosic substrate) using free and immobilised enzymes. Cellulase from Trichoderma reesei was immobilised on an activated magnetic support by covalent binding and its activity was compared with that of the free enzyme to hydrolyse microcrystalline cellulose and hemp hurds on the basis of thermostability and reusability.

Bead milling for lipid recovery from thraustochytrid cells and selective hydrolysis of Schizochytrium DT3 oil using lipase

Marine microalgae present a renewable alternative source for sustainable production of omega-3 fatty acids, as compared to conventional sources such as krill oil and fish oil. In this study, we optimised a method for lipid extraction from marine thraustochytrids using a bead mill and enzymatic concentration of omega-3 fatty acids from the thraustochytrid oil. The optimised lipid extraction conditions were, bead size 0.4-0.6μm, 4500rpm, 4min of processing time at 5g biomass concentration. The maximum lipid yield (% dry weight basis) achieved at optimum conditions were 40.5% for Schizochytrium sp. S31 (ATCC) and 49.4% for Schizochytrium sp. DT3 (in-house isolate). DT3 oil contained 39.8% docosahexaenoic acid (DHA) as a percentage of lipid, a higher DHA percentage than S31. Partial hydrolysis of DT3 oil using Candida rugosa lipase was performed to enrich omega-3 polyunsaturated fatty acids (PUFAs) in the glyceride portion. Total omega-3 fatty acid content was increased to 88.7%.

Understanding physicochemical changes in pretreated and enzyme hydrolysed hemp (Cannabis sativa) biomass for biorefinery development

The physicochemical properties of hemp biomass structure to pretreatment and enzymatic hydrolysis were investigated to improve upon reducing sugar production for biofuel development. Sodium hydroxide pretreated biomass (SHPB) yielded maximum conversion of holocellulose into reducing sugar (72 %). Scanning electron microscopy (SEM) revealed that enzymatic hydrolysis generated regular micropores in the fragmented biomass structure. The thermogravimetric analysis (TGA) curve suggested the degradation of hemicellulose and cellulose, which conformed well to the subsequent nuclear magnetic resonance (NMR) studies indicating the presence of α- and β-glucose (28.4 %) and α- and β-xylose (10.7 %), the major carbohydrate components commonly found in hydrolysis products of hemicellulose and cellulose. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra showed stretching modes of the lignin acetyl group, suggesting the loosening of the polymer matrix and thus the exposure of the cellulose polymorphs. X-ray diffraction pattern indicated that enzymatic hydrolysis caused a higher crystallinity index (36.71), due to the fragmentation of amorphous cellulose leading to the reducing sugar production suitable for biofuel development.

α,ω-Bis(trichlorostannyl)alkanes: unravelling the hydrolysis pathway to organotin-oxo oligomers

New α,ω-bis(trichlorostannyl)alkanes, Cl₃Sn(CH₂)_nSnCl₃ [n = 3-5, 8], have been synthesized via tin-phenyl bond cleavage reactions on α,ω-bis(triphenylstannyl)alkanes, Ph₃Sn(CH₂)_nSnPh₃ [n = 3-5, 8], using either SnCl₄ or concentrated hydrochloric acid. Some key missing links, (H₂O)Cl₃Sn(CH₂)₃SnCl₃(H₂O) (1a) and (H₂O)₂Cl₃Sn(CH₂)₃SnCl₃(H₂O)₂ (6), in the hydrolysis pathway of organotin trichlorides were identified. Crystal structures of the nonassociated di-tin compounds (H₂O)Cl₃Sn(CH₂)₃SnCl₃(H₂O) (1a) and (H₂O)₂Cl₃Sn(CH₂)₃SnCl₃(H₂O)₂ (6, isolated as the 18-crown-6 cocrystal acetonitrile solvate) as well as the polymeric hydrolysis product [H₂O(OH)Cl₂Sn(CH₂)₃SnCl₂(OH)H₂O·₂H2O]_n (7·2H₂O) are reported.

Hydrolysis of dinuclear spacer-bridged diorganotin(IV) triflates. A novel cationic double ladder with supramolecular association

A series of oligomethylene-bridged diorganotin triflates R(OTf)₂Sn(CH₂)_nSn(OTf)₂R (R = CH₂SiMe₃; n = 3, 4, 8, 10) were synthesized by reaction of triflic acid with the precursor oxides R(O)Sn(CH₂)_nSn(O)R. On the basis of ¹¹⁹Sn NMR (in acetonitrile) the triflates appear to be the simple six-coordinated ionic species [(MeCN)₄(RSn(CH₂)_nSnR)(MeCN)₄]²⁺. These triflates readily undergo hydrolysis to give products, the identity of which depends on the length of the oligomethylene bridge. For n = 3 (5), the solid-state structure shows association of two dimeric units, which results in a tetracationic double ladder. Extensive hydrogen bonding gives rise to a supramolecular association. Solution ¹¹⁹Sn NMR and ES MS suggest some dissociation of 5 into dimers containing four tin atoms and possibly monomers containing two tin atoms. A rudimentary solid-state structure for n = 4 (6) indicates a linear polymer based on dimeric (four tin atoms) units. The structure of 6 also features extensive hydrogen bonding, this time effectively giving rise to alternating layers of cations and anions.

Hydrolysis of (Me3SiCH2)PhSnCl2. Isomerisation of the dimeric tetraorganodistannoxane [(Me3SiCH2)Ph(Cl)SnOSn(Cl)-Ph(CH2SiMe3)]2

The hydrolysis of (Me₃SiCH₂)PhSnCl₂( 1) was studied under two different reaction conditions (i) by using an excess of aqueous NaOH in toluene at reflux temperature and (ii) by using small amounts of NEt₃ and water in CH₂Cl₂ at room temperature. For (i) the products  (Me₃SiCH₂)Ph₂SnOSnPh₂(CH₂SiMe₃)( 2) and [(Me₃SiCH₂Sn)₁₂O₁₄(OH)₆](OH)₂( 3) were isolated indicating that a phenyl group migration took place. For (ii) the dimeric tetraorganodistannoxane [(Me₃SiCH₂)Ph(Cl)SnOSn(Cl)Ph(CH₂SiMe₃)]₂( 4) was obtained. In solution, 4 exists as an equilibrium mixture of all five possible isomers 4a–4e; in the solid state two of these isomers 4d and 4e co-crystallized in the same crystal modification. The observation of interconvertible isomers of 4 was attributed to the kinetic lability of the ladder-like Sn4O2Cl4 structural motif. Compounds 1 and 4 were investigated by X-ray crystallography.

Estimates of Heterozostera tasmanica, Zostera muelleri and Ruppia megacarpa distribution and biomass in the Hopkins Estuary, western Victoria, by GIS

Knowledge of the spatial arrangement of the seagrass distribution and biomass within the Hopkins Estuary is an essential step towards gaining an understanding of the functioning of the estuarine ecosystem. This study marks the first attempt to map seagrass distribution and model seagrass biomass and epiphyte biomass along depth gradients by the use of global positioning system (GPS) and geographical information system (GIS) technologies in the estuary. For mapping seagrass in small estuaries, ground-surveying the entire system is feasible. Three species of seagrasses, Heterozostera tasmanica (Martens ex Aschers), Zostera muelleri (Irmisch ex Aschers) and Ruppia megacarpa (Mason), were identified in the Hopkins Estuary. All beds investigated contained a mixed species relationship. Three harvest techniques were trialed in a pilot study, with the 25 × 25-cm quadrat statistically most appropriate. Biomass of seagrasses and epiphytes was found to vary significantly with depth, but not between sites. The average estimate of biomass for total seagrasses and their epiphytes in the estuary in January 2000 was 222.7 g m^–2 (dry weight). Of the total biomass, 50.6% or 112.7 g m^–2 (dry weight) was contributed by seagrasses and 49.4% of the biomass (110.0 g m^–2) were epiphytes. Of the 50.6% of the total biomass represented by seagrasses, 39.3% (87.5 g m^–2) were leaves and 11.3% (25.2 g m^–2) were rhizomes. The total area of seagrasses present in the Hopkins Estuary was estimated to be 0.4 ± 0.005 km², with the total area of the estuary estimated to be 1.6 ± 0.02 km² (25% cover). The total standing crop of seagrasses and epiphytes in the Hopkins Estuary in January 2000 was estimated to be 102.3 ± 57 t in dry weight, 56% (56.9 ± 17 t, dry weight) seagrasses and 44% (45.4 ± 19 t, dry weight) epiphytes. Of the seagrass biomass, 39% (39.7 ± 13 t, dry weight) was contributed by leaves and 17% (17.3 ± 7 t, dry weight) by rhizomes.

Hydrolysis of bis((trimethylsilyl)methyl)tin dihalides. Crystallographic and spectroscopic study of the hydrolysis pathway

The synthesis and characterization by multinuclear NMR spectroscopy of the diorganotin dihalides (Me₃SiCH₂)₂SnX₂ (1, X = Cl; 2, X = Br), the diorganotin dichloride water adduct (Me₃SiCH₂)₂SnCl₂·H₂O (1a), the dimeric tetraorganodistannoxanes [(Me₃SiCH₂)₂(X)SnOSn(Y)(CH₂SiMe₃)₂]₂ (3, X = Y = Cl; 4, X = Br, Y = OH; 5, X = Br, Y = F; 6, X = Y = OH; 8, X = Cl, Y = OH), and the molecular diorganotin oxide cyclo-[(Me₃SiCH₂)₂SnO]₃ (7) are reported. The structures in the solid state of compounds 1a, 3, 6, and 7 were determined by single-crystal X-ray analysis. In toluene solution, the hydroxy-substituted tetraorganodistannoxane 6 is in equilibrium with the diorganotin oxide 7 and water. The eight-membered diorganotin oxide cyclo-[(Me₃SiCH₂)₂SnO]₄ (7a) is proposed to be involved in this equilibrium. On the basis of the results of this and previous works, a general hydrolysis pathway is developed for diorganotin dichlorides containing reasonably bulky substituents.

Biomass and energy transfer to baleen whales in the South Atlantic sector of the Southern Ocean

Baleen whales are an important group of predators on Antarctic krill in the Southern Ocean. During the CCAMLR 2000 Survey to estimate the biomass and distribution of Antarctic krill, International Whaling Commission observers carried out a visual line transect survey to estimate the number of baleen whales occurring in the survey area. This paper reviews techniques used to estimate krill consumption by baleen whales and in combination with estimates of whale abundance estimates of krill consumption are generated for the South Atlantic sector of the Southern Ocean. This survey estimates that the present populations of whales feeding in this region are likely to consume approximately 1.6 million tonnes, but possibly up to as much as 2.7 million tonnes of krill within the summer season. Although this only represents 4–6% of the estimated krill biomass in the region (and probably less than this percentage of the total annual krill production), the depleted numbers of baleen whales resulting from past or current whaling activities should be taken into account when setting quotas for the commercial exploitation of krill if there is to be a recovery to pre-exploitation biomass levels of baleen whales.

Cell disruption optimization and covalent immobilization of beta-D-galactosidase from kluyveromyces marxianus YW-1 for lactose hydrolysis in milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.

Development of a stable continuous flow immobilized enzyme reactor for the hydrolysis of inulin

A 23.5-fold purified exoinulinase with a specific activity of 413 IU/mg and covalently immobilized on Duolite A568 has been used for the development of a continuous flow immobilized enzyme reactor for the hydrolysis of inulin. In a packed bed reactor containing 72 IU of exoinulinase from Kluyveromyces marxianus YS-1, inulin solution (5%, pH 5.5) with a flow rate of 4 mL/h was completely hydrolyzed at 55 °C. The reactor was run continuously for 75 days and its experimental half-life was 72 days under the optimized operational conditions. The volumetric productivity and fructose yield of the reactor were 44.5 g reducing sugars/L/h and 53.3 g/L, respectively. The hydrolyzed product was a mixture of fructose (95.8%) and glucose (4.2%) having an average fructose/glucose ratio of 24. An attempt has also been made to substitute pure inulin with raw Asparagus racemosus inulin to determine the operational stability of the developed reactor. The system remained operational only for 11 days, where 85.9% hydrolysis of raw inulin was achieved.

One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.