Samuel R. A. Collins

High concentrations of cellulosic ethanol achieved by fed batch semi simultaneous saccharification and fermentation of waste-paper

Highlights ► Batch addition of paper waste in SSSF results in up to 11.6% (v/v) ethanol. ► Low overall enzyme loadings (3.7 FPU/g substrate). ► High cumulative substrate loadings (65% w/v). ► High ethanol concentrations will improve distillation efficiencies.

Characterization of cell wall components of wheat straw following hydrothermal pretreatment and fractionation

Thermophysical pretreatment enhances the enzymatic hydrolysis of lignocellulose. However, its impact on cell wall chemistry is still poorly understood. This paper reports the effects of hydrothermal pretreatment on the degradation and alkali-extractability of wheat straw cell wall polymers. Pretreatment resulted in loss and/or solubilization of arabinoxylans (by 53%), ferulic and diferulic acids which are important cross-linking agents accompanied by concomitant increases in cellulose (up to 43%) and lignin (29%). The remaining water-insoluble hemicelluloses were more readily extractable in alkali and were reduced in molecular weight indicating substantial thermochemical depolymerization. They were also associated with smaller but significant amounts of (cellulose-derived) glucose. The alkali-insoluble residues consisted predominantly of cellulosic glucose and lignin and contained p-coumaric acid. The depolymerization of hemicelluloses, reduction in cinnamic acids and partial degradation of cellulose is likely to contribute significantly to the accessibility of cellulases during subsequent enzymolysis.

Spontaneous Mutation Reveals Influence of Exopolysaccharide on Lactobacillus johnsonii Surface Characteristics

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

Characterization of cell wall components of wheat bran following hydrothermal pretreatment and fractionation.

BackgroundPretreatments are a prerequisite for enzymatic hydrolysis of biomass and production of ethanol. They are considered to open up the plant cell wall structure by altering, moving or solubilizing lignin and hydrolyzing a proportion of hemicellulosic moieties. However, there is little information concerning pretreatment-induced changes on wheat bran cell wall polymers and indeed on changes in cell wall phenolic esters in bran or other lignocellulosic biomass. Here, we evaluate polymeric changes (chemical and physical) as a result of selected hydrothermal pretreatment conditions on destarched wheat bran using controlled polymer extraction methods. Quantification of cell wall components together with soluble oligosaccharides, the insoluble residues and ease of extractability and fractionation of biomass residues were conducted.ResultsPretreatment solubilized selected arabinoxylans and associated cross-linking ferulic and diferulic acids with a concomitant increase in lignin and cellulosic glucose. The remaining insoluble arabinoxylans were more readily extractable in alkali and showed considerable depolymerization. The degree of arabinose substitution was less in xylans released by higher concentrations of alkali. The recalcitrant biomass which remained after pretreatment and alkali extraction contained mostly cellulosic glucose and Klason lignin. Pretreatment generated small but insignificant amounts of yeast-inhibiting compounds such as furfural and hydroxymethyl furfural.As such, simultaneous saccharification and fermentation of the hydrothermally pretreated bran resulted in increased ethanol yields compared to that of the control (97.5% compared to 63% theoretical).ConclusionHydrothermal pretreatment of destarched wheat bran resulted in degradation and depolymerization of the hemicellulosic arabinoxylans together with some breakdown of cellulosic glucose. This was accompanied by a significant reduction in the cross-linking phenolic acids such as ferulic and diferulic acids. The results suggest that hydrothermal pretreatment enhances enzymatic digestibility of the cellulose not only by depolymerization and solubilization of the hemicelluloses but by breakdown of interpolymeric phenolic cross-links between the remaining insoluble polymers. This allows easier access of hydrolytic enzymes by opening or loosening of the cell wall thus resulting in enhanced saccharification of cellulose and subsequent fermentation to ethanol. The reduction in cinnamic acids by selected breeding or biotechnological approaches could provide a useful basis for improved saccharification and fractionation of wheat bran polysaccharides.

Variation in the chemical composition of wheat straw: the role of tissue ratio and composition

BackgroundWheat straw is an attractive substrate for second generation ethanol production because it will complement and augment wheat production rather than competing with food production. However, like other sources of lignocellulosic biomass, even from a single species, it is heterogeneous in nature due to the different tissues and cell types, and this has implications for saccharification efficiency. The aim of this study has been to use Fourier transform infrared (FTIR) spectroscopy and Partial least squares (PLS) modelling to rapidly screen wheat cultivars for the levels of component tissues, the carbohydrate composition and lignin content, and the levels of simple cross-linking phenolics such as ferulic and diferulic acids.ResultsFTIR spectroscopy and PLS modelling was used to analyze the tissue and chemical composition of wheat straw biomass. Predictive models were developed to evaluate the variability in the concentrations of the cell wall sugars, cell wall phenolics and acid-insoluble lignin. Models for the main sugars, phenolics and lignin were validated and then used to evaluate the variation in total biomass composition across 90 cultivars of wheat grown over two seasons.ConclusionsWhilst carbohydrate and lignin components varied across the varieties, this mainly reflected differences in the ratios of the component tissues rather than differences in the composition of those tissues. Further analysis indicated that on a mol% basis, relative levels of sugars within the tissues varied to only a small degree. There were no clear associations between simple phenolics and tissues. The results provide a basis for improving biomass quality for biofuels production through selection of cultivars with appropriate tissue ratios.

Chemical characterisation and analysis of the cell wall polysaccharides of duckweed (Lemna minor).

Duckweed is potentially an ideal biofuel feedstock due to its high proportion of cellulose and starch and low lignin content. However, there is little detailed information on the composition and structure of duckweed cell walls relevant to optimising the conversion of duckweed biomass to ethanol and other biorefinery products. This study reports that, for the variety and batch evaluated, carbohydrates constitute 51.2% (w/w) of dry matter while starch accounts for 19.9%. This study, for the first time, analyses duckweed cell wall composition through a detailed sequential extraction. The cell wall is rich in cellulose and also contains 20.3% pectin comprising galacturonan, xylogalacturonan, rhamnogalacturonan; 3.5% hemicellulose comprising xyloglucan and xylan, and 0.03% phenolics. In addition, essential fatty acids (0.6%, α-linolenic and linoleic/linoelaidic acid) and p-coumaric acid (0.015%) respectively are the most abundant fatty acids and phenolics in whole duckweed.

Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation.

Highlights • Steam explosion of copier paper reduces xylose and produces inhibitors.• Steam explosion at SF 3.6 and 3.9 increased initial rates of saccharification.• Steam explosion at moderate severities may reduce processing times.• Co-steam explosion of waste paper and wheat straw reduces inhibitor production.

Enzyme-aided Fractionation of Brewer's Spent Grains in Pilot Scale

Brewers spent grain (BSG) is an important coproduct of the brewing industry and is generally used in animal feed. Recently, there has been considerable research into the use of enzymes to convert BSG into more value-added products. In this study, the efficiency of enzymatic fractionation of freshly produced BSG was studied in pilot scale. Carbohydrate-and protein-degrading enzymes were applied sequentially to produce three fractions: carbohydrate hydrolysate, protein hydrolysate, and a solid lignin-carbohydrate residue. To minimize microbial contamination, processing was performed at 60°C for 4 hr. In all, 14 and 36% of the original dry matter was liberated by carbohydrase and protease, respectively. The carbohydrate-degrading enzyme cocktail produced cellulose- and hemicellulose-derived mono- and oligosaccharides. The protease treatment released peptide-enriched hydrolysate. Approximately half of the original BSG solids were solubilized during the two-stage enzymatic process. Although the protease efficiently released the majority of BSG proteins, the carbohydrate matrix was much less accessible to enzyme attack. The impact of scale-up to pilot scale was compared with previous laboratory-scale studies, and the results were found to be in good agreement.

Optimising conditions for bioethanol production from rice husk and rice straw: effects of pre-treatment on liquor composition and fermentation inhibitors

BackgroundRice straw and husk are globally significant sources of cellulose-rich biomass and there is great interest in converting them to bioethanol. However, rice husk is reportedly much more recalcitrant than rice straw and produces larger quantities of fermentation inhibitors. The aim of this study was to explore the underlying differences between rice straw and rice husk with reference to the composition of the pre-treatment liquors and their impacts on saccharification and fermentation. This has been carried out by developing quantitative NMR screening methods.ResultsAir-dried rice husk and rice straw from the same cultivar were used as substrates. Carbohydrate compositions were similar, whereas lignin contents differed significantly (husk: 35.3% w/w of raw material; straw 22.1% w/w of raw material). Substrates were hydrothermally pre-treated with high-pressure microwave processing across a wide range of severities. 25 compounds were identified from the liquors of both pre-treated rice husk and rice straw. However, the quantities of compounds differed between the two substrates. Fermentation inhibitors such as 5-HMF and 2-FA were highest in husk liquors, and formic acid was higher in straw liquors. At a pre-treatment severity of 3.65, twice as much ethanol was produced from rice straw (14.22% dry weight of substrate) compared with the yield from rice husk (7.55% dry weight of substrate). Above severities of 5, fermentation was inhibited in both straw and husk. In addition to inhibitors, high levels of cellulase-inhibiting xylo-oligomers and xylose were found and at much higher concentrations in rice husk liquor. At low severities, organic acids and related intracellular metabolites were released into the liquor.ConclusionsRice husk recalcitrance to saccharification is probably due to the much higher levels of lignin and, from other studies, likely high levels of silica. Therefore, if highly polluting chemical pre-treatments and multi-step biorefining processes are to be avoided, rice husk may need to be improved through selective breeding strategies, although more careful control of pre-treatment may be sufficient to reduce the levels of fermentation inhibitors, e.g. through steam explosion-induced volatilisation. For rice straw, pre-treating at severities of between 3.65 and 4.25 would give a glucose yield of between 37.5 and 40% (w/DW, dry weight of the substrate) close to the theoretical yield of 44.1% w/DW, and an insignificant yield of total inhibitors.

Yeast diversity in relation to the production of fuels and chemicals

In addition to ethanol, yeasts have the potential to produce many other industrially-relevant chemicals from numerous different carbon sources. However there remains a paucity of information about overall capability across the yeast family tree. Here, 11 diverse species of yeasts with genetic backgrounds representative of different branches of the family tree were investigated. They were compared for their abilities to grow on a range of sugar carbon sources, to produce potential platform chemicals from such substrates and to ferment hydrothermally pretreated rice straw under simultaneous saccharification and fermentation conditions. The yeasts differed considerably in their metabolic capabilities and production of ethanol. A number could produce significant amounts of ethyl acetate, arabinitol, glycerol and acetate in addition to ethanol, including from hitherto unreported carbon sources. They also demonstrated widely differing efficiencies in the fermentation of sugars derived from pre-treated rice straw biomass and differential sensitivities to fermentation inhibitors. A new catabolic property of Rhodotorula mucilaginosa (NCYC 65) was discovered in which sugar substrate is cleaved but the products are not metabolised. We propose that engineering this and some of the other properties discovered in this study and transferring such properties to conventional industrial yeast strains could greatly expand their biotechnological utility.