Bacterial biodegradation of extractives and patterns of bordered pit membrane attack in pine wood

Wood extractives, commonly referred to as pitch, cause major problems in the manufacturing of pulp and paper. Treatment of nonsterile southern yellow pine chips for 14 days with Pseudomonas fluorescens, Pseudomonas sp., Xanthomonas campestris, and Serratia marcescens reduced wood extractives by as much as 40%. Control treatments receiving only water lost 11% of extractives due to the growth of naturally occurring microorganisms. Control treatments were visually discolored after the 14-day incubation, whereas bacterium-treated wood chips were free of dark staining. Investigations using P. fluorescens NRRL B21432 showed that all individual resin and fatty acid components of the pine wood extractives were substantially reduced. Micromorphological observations showed that bacteria were able to colonize resin canals, ray parenchyma cells, and tracheids. Tracheid pit membranes within bordered pit chambers were degraded after treatment with P. fluorescens NRRL B21432. P. fluorescens and the other bacteria tested appear to have the potential for biological processing to substantially reduce wood extractives in pine wood chips prior to the paper making process so that problems associated with pitch in pulp mills can be controlled.     

The influence of iron,sulphite and paraformaldehyde on the formation of 2.3 butanediol in the cultures of bacillus polymyxa

Summary InB. polymyxa cultures containing sucrose as the substrate the amount of 2.3 butanediol decreases between 10 and 30 days of fermentation but after 30 days, the formation of the glycol increases. If iron or sulphite ions are present in the culture ofB. polymyxa the yield of 2.3 butanediol is small in the first 10 days, as compared to the control culture but it gradually increases with the increase of the period of fermentation. This increase was observed upto 50 days. In the culture containing paraformaldehyde 2.3 butanediol is not formed. The sugar consumption in the cultures is high only during the first ten days of fermentation and after that though there is enough sugar present, it is not utilised and the amount of 2.3 butanediol goes on increasing. It appears that 2.3 butanediol is formed from some intermediate material formed during the period of high sugar consumption. Later the increase of 2.3 butanediol is due to its formation from the intermediate compound.     

Comparison of aromatic monomers in lignocellulosic biomass prehydrolysates

Differences in the relative toxicity of xylose-rich prehydrolysates derived from woody and herbaceous feedstocks are likely due to the relative abundance of a variety of inhibitory compounds. Acetate, as well as several aromatic monomers, has been shown to be an inhibitor of the xylose-fermenting yeast, Pichia stipitis. Comparative information on the concentration of known and likely inhibitors, other than acetate, is lacking. The present study provides data on the aromatic monomer composition of representative herbaceous and woody prehydrolysates. Dilute-acid prehydrolysates were prepared from three feedstocks; two herbaceous, corn stover and switchgrass (Panicum virgatum L.), and one woody (poplar). The prehydrolysates were neutralized with Ca(OH)₂ extracted with ethyl acetate, trimethylsilylated, and analyzed by GC-MS. Fourteen aromatic monomers were tentatively identified by comparison with published mass spectra. The concentrations of the aromatic monomers totalled 112, 141 and 247 mg L⁻¹ for corn stover, switchgrass and poplar prehydrolysates, respectively. This is also the order of increasing inhibition of growth and ethanol productivity observed for Pichia fermentations. The woody prehydrolysate contained approximately four-fold more syringyl-based monomers than did the herbaceous prehydrolysates, while guaiacyl-containing compounds were more evenly distributed. Received 24 April 1998/ Accepted in revised form 8 June 1998     

Fermentation of Xylan,Corn Fiber,or Sugars to Acetoin and Butanediol by Bacillus polymyxa Strains

Bacillus polymyxa can produce levo-butanediol, a potential biogradable anti-freeze, and ethanol, a fuel additive, using starch-based fermentations. To explore use of less expensive biomass fermentation substrates, we screened B. polymyxa strains for good growth on xylans. During aerobic growth on glucose, six selected xylanolytic strains produced mainly acetoin and butanediol plus lesser amounts of acetaldehyde and ethanol. Undesirable acetoin formation was eliminated by anaerobic growth on glucose, but substrate usage, butanediol, and other fermentation products were greatly reduced. High xylanase activity occurred with growth on xylans or corn fiber, and about 50–65% of oatspelt xylan and 25–35% of the corn fiber were used during aerobic growth, but unexpectedly no butanediol and only small levels of acetoin were produced. Aerobic growth on arabinose, arabinose plus glucose, or xylose plus glucose resulted in both acetoin and butanediol formation. Little or no butanediol was made from xylose alone. Growth on an acid hydrolysate of corn fiber that contained a mixture of these sugars resulted in the formation of acetoin, acetaldehyde, and ethanol, but very little butanediol. The data suggest B. polymyxa is limited in conversion of xylan-rich biomass sources or their hydrolysates to butanediol. This limitation might be overcome by using better cultivation conditions and/or genetically engineered strains.     

A study of the influence of zinc ions on the production of 2,3 butanediol by Pichia indica

Summary Pichia indica produces 2,3 butanediol during fermentation. The formation of this glycol increases with the presence of Zn⁺⁺ in the culture. The increase of Zn⁺⁺ concentration increases the formation of 2,3 butanediol in the culture till a maximum is reached. Further increase of Zn⁺⁺ concentration decreases the diol production. On increasing the period of fermentation the glycol disappears from the culture.     

The combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

Summary Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50–60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50° C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30° C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20–40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4–0.5 g/g of reducing sugar utilized.     

Butanediol production from lignocellulosic substrates by Klebsiella pneumoniae grown in sequential co-culture with Clostridium thermocellum

Summary A sequential co-culture approach was investigated for the conversion of lignocellulosic substrates to butanediol and ethanol. Growth of Clostridium thermocellum on solka floc and aspenwood xylan resulted in the release of extracellular endoglucanase and xylanase enzymes into the culture medium. Low levels of fermentation products were formed and unutilized sugars accumulated in the medium. Inoculation of Klebsiella pneumoniae as a sequential culture resulted in the rapid utilization of the accumulated sugars and the formation of additional fermentation products, including butanediol, ethanol, and acetoin. This approach was applicable to the use of mixed cellulose and hemicellulose substrates, including steam-exploded aspenwood. Further improvement in solvent production from steam-exploded substrates could be obtained by using a fed-batch approach to circumvent the problem of inhibitors associated with the natural substrates.     

Natural durability of selected larch and Scots pine heartwoods in laboratory and field tests

The aim of this study was to compare natural durability of Siberian larch heartwood grown in Siberia and Sweden as well as European larch and Scots pine heartwood grown in Sweden. The study was based on standard in- and above ground tests lasting 12 years but laboratory decay tests with white and brown rot fungi was also included. Field test results showed that Siberian larch heartwood from Siberia was the most durable among the studied heartwoods with a decay index of 60 after 12 years in Simlångsdalen (Sweden), while European larch heartwood grown in Sweden, was decayed to failure before the end of the test. Scots pine heartwood was found to perform similarly to Siberian larch from Siberia. No relationship could be established between natural durability of examined heartwoods and their water absorption behavior; however, strong correlation to the total amount of extractives was observed. Scots pine and Siberian larch heartwood from Siberia had 12.7 and 19.6% total extractives content respectively but the extractives composition differs. The study revealed also that lignin and monosaccharide content could not explain the variations in decay resistance of the studied heartwoods. No similarities in the natural durability revealed by laboratory and field tests were observed.     

Simulating the impacts of southern pine beetle and fire on the dynamics of xerophytic pine landscapes in the southern Appalachians

Question: Can fire be used to maintain Yellow pine (Pinus subgenus Diploxylon) stands disturbed by periodic outbreaks of southern pine beetle? Location: Southern Appalachian Mountains, USA. Methods: We used LANDIS to model vegetation disturbance and succession on four grids representative of xeric landscapes in the southern Appalachians. Forest dynamics of each landscape were simulated under three disturbance scenarios: southern pine beetle, fire, and southern pine beetle and fire, as well as a no disturbance scenario. We compared trends in the abundance of pine and hardwood functional types as well as individual species. Results: Yellow pine abundance and open woodland conditions were best maintained by a combination of fire and southern pine beetle disturbance on both low elevation sites as well as mid‐elevation ridges & peaks. On mid‐elevation SE‐W facing slopes, pine woodlands were best maintained by fire alone. Conclusions: Our simulations suggest that fire can help maintain open pine woodlands in stands affected by southern pine beetle outbreaks.     

Chloroplast DNA Transgresses Species Boundaries and Evolves at Variable Rates in the California Closed-Cone Pines (Pinus radiata, P. muricata, and P. attenuata)

We studied phylogenetic relationships among populations and species in the California closed-cone pines (Pinus radiata D. Don, P. attenuata Lemm., and P. muricata D. Don) via chloroplast DNA restriction site analysis. Data on genetic polymorphism within and among 19 populations in the three species were collected using9 to 20 restriction enzymes and 38 to 384 trees. Because only five clades and extremely low intraclade diversity were found, additional phylogenetic data were collected using a single representative per clade and two outgroup species, P. oocarpa Schiede and P. jeffreyi Loud. In total, 25 restriction enzymes were employed and approximately 2.7 kb surveyed (2.3% of genome). The five clades recognized were Monterey pine, knob-cone pine, and the southern, intermediate, and northern races of bishop pine. On the basis of bootstrapping, both Wagner and Dollo parsimony analyses strongly separated the northern and intermediate races of bishop pine from the southern race; knobcone pine from Monterey and bishop pines; and the closed-cone pines from the two outgroups. Approximate divergence times were estimated for the lineages leading to knob-cone pine and to the intermediate and northern populations of bishop pine. The position of Monterey pine relative to bishop pine within their monophyletic clade was unresolved. Surprisingly, Montery pine and the southern race of bishop pine were much more similar to one another than was the southern race of bishop pine to its conspecific intermediate and northern races. Both the Monterey and southern bishop pine lineages also evolved severalfold more slowly than did the knobcone pine and intermediate-northern bishop pine lineages. These results differ significantly from a recent allozyme study, corroborating previous observations that chloroplast genome phylogeny can depart substantially from that of nuclear genes.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid production.     

Genetic improvement of the chemical composition of Scots pine (Pinus sylvestris L.) juvenile wood for bioenergy production

Chemical composition is one of the key characteristics that determines wood quality and in turn its suitability for different end products and applications. The inclusion of chemical compositional traits in forest tree improvement requires high‐throughput techniques capable of rapid, non‐destructive and cost‐efficient assessment of large‐scale breeding experiments. We tested whether Fourier‐transform infrared (FTIR) spectroscopy, coupled with partial least squares regression, could serve as an alternative to traditional wet chemistry protocols for the determination of the chemical composition of juvenile wood in Scots pine for tree improvement purposes. FTIR spectra were acquired for 1,245 trees selected in two Scots pine (Pinus sylvestris L.) full‐sib progeny tests located in northern Sweden. Predictive models were developed using 70 reference samples with known chemical composition (the proportion of lignin, carbohydrates [cellulose, hemicelluloses and their structural monosaccharides glucose, mannose, xylose, galactose, and arabinose] and extractives). Individual‐tree narrow‐sense heritabilities and additive genetic correlations were estimated for all chemical traits as well as for growth (height and stem diameter) and wood quality traits (density and stiffness). Genetic control of the chemical traits was mostly moderate. Of the major chemical components, highest heritabilities were observed for hemicelluloses (0.43–0.47), intermediate for lignin and extractives (0.30–0.39), and lowest for cellulose (0.20–0.25). Additive genetic correlations among chemical traits were, except for extractives, positive while those between chemical and wood quality traits were negative. In both groups (chemical and wood quality traits), correlations with extractives exhibited opposite signs. Correlations of chemical traits with growth traits were near zero. The best strategy for genetic improvement of Scots pine juvenile wood for bioenergy production is to decrease and stabilize the content of extractives among trees and then focus on increasing the cellulose:lignin ratio.     

The effect of water soluble Scots pine (Pinus sylvestris L.) and Sitka spruce [Picea sitchensis (Bong.) carr.] heartwood and sapwood extracts on the growth of selected Trichoderma species

Extractable materials from some timber species have been identified which prevent wood decay; however, little has been reported on the effect(s) of such materials against mould species that colonize timber. With increasing interest in the use of Trichoderma species, both as agents of permeability enhancement and biological control, more information is required on how chemical components within fresh and processed timber influence growth of Trichoderma. Fresh and dried samples of Scots pine and Sitka spruce sapwood and heartwood were leached in a Soxhlet apparatus and the resulting extract was combined with malt extract agar and inoculated with Trichoderma. Trichoderma isolates were inhibited to varying degrees by extractives removed from fresh and dried heartwood of the two timbers. Growth on sapwood extractives, however, showed a lesser degree of inhibition. The implications of the results for applications of Trichoderma in timber are discussed.     

Suitability of aspenwood biologically delignified with Pheblia tremellosus for fermentation to ethanol or butanediol

Summary Enzymatic conversion of lignocellulosic material to fuels and chemicals depends on a initial pretreatment to render the cellulose more susceptible to enzymatic attack. Biological delignification of aspenwood with the fungus Phlebia tremellosus was compared to steaming as a pretreatment method.The biologically delignified aspenwood (BDA) had a high pentosan content and did not contain inhibitors of enzymatic hydrolysis or subsequent fermentation. In contrast, the steamed aspenwood required a water extraction step to remove the inhibitory material and this step also removed most of the pentosan. The yield of treated material was 90% from biological delignification and 70% from steaming.The cellulose in the BDA was less accessible to the cellulase enzymes than the steamed aspenwood. Combined hydrolysis and fermentation with Saccharomyces cerevisiae gave a lower yield of ethanol from BDA than from the steamed aspenwood, but the yields based on the weight of substrate before pretreatment were comparable. Combined hydrolysis and fermentation with Klebsiella pneumoniae gave higher yields of butanediol from BDA than from steamed aspenwood, because Klebsiella can ferment the xylose which was present in the biologically treated aspenwood. Trichoderma harzianum produced lower levels of cellulase enzymes when grown on BDA than when grown on steamed aspenwood and this was related to the xylan found in the biologically treated material.Abbreviations BDA biologically delignified aspenwood - SEA-WI steam-exploded, water-extracted aspenwood - AI-SEA-WI acid-impregnated, steam-exploded, water-extracted aspenwood - CHF combined hydrolysis and fermentation - FP filter paper     

A comparative study of the enzymatic hydrolysis of acid-pretreated white pine and mixed hardwood

Removal of hemicellulose by acid pretreatment in a flow reactor followed by enzymatic hydrolysis of the neutralized slurry has resulted in glucose yields as high as 95% for mixed hardwood. For white pine, however, the maximum glucose yield is 65%. Although pine has a higher extractives content, removal of the extractives prior to enzymatic hydrolysis does not increases the glucose yield. Pore size measurements reveal that the increase in pore volume, in the size range of the cellulase molecule, following pretreatment for pine is only about one-half the value obtained with mixed hardwood. This suggests that pore volume is an important determinant of substrate-enzyme reactivity.     

Red oak wood derived inhibitors in the ethanol fermentation of xylose byPichia stipitis CBS 5776

Summary Xylose, the predominant sugar in red oak prehydrolysate, is fermented to ethanol byPichia stipitis CBS 5776. Toxic model compounds derived from red oak hemicelluloses, lignin, and extractives inhibited the fermentation. Treatment of the prehydrolysate with molecular sieve and mixed bed ion resins facilitated the ethanol fermentation giving about 10 g/l ethanol from 32 g/l initial xylose. Fermentation inhibitors derived from red oak lignin and extractives were identified.     

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3‐butanediol by Paenibacillus polymyxa CJX518

The introduction of an NADH/NAD⁺ regeneration system can regulate the distribution between acetoin and 2,3‐butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB₂) was used to regulate the NADH/NAD⁺ ratio and, consequently, the distribution of acetoin and 2,3‐butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed‐batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB₂ production of recombinant E. coli could maintain a relatively low NADH/NAD⁺ ratio by changing NADH dehydrogenase activity. It was also found that 2,3‐butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB₂ or by being in the artificial consortium that produces VB₂. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD⁺ ratio to improve the acetoin production of P. polymyxa.     

The bioconversion of wood hydrolyzates to butanol and butanediol

Steam-exploded aspenwood chips were acid hydrolysed to their component sugars. Near theoretical solvent yields were achieved in both the acetone-butanol-ethanol (ABE) fermentation and 2,3-butanediol fermentation of these liberated sugars. When Clostridium acetobutylicum was grown on wood hydrolysates, final butanol yields of 9.0 g/L (0.26 g of butanol per g of sugar consumed) were obtained. When Klebsiella pneumoniae was grown on the wood hydrolysates, final butanediol concentrations exceeded 20 g/L, resulting in a bioconversion efficiency approaching 0.5 g of butanediol per g of sugar utilised.     

Resistance of Scots pine wood to Brown-rot fungi after long-term forest fertilization

The susceptibility of Scots pine (Pinus sylvestris L.) sap- and heartwood against the wood decaying brown-rot fungus (Coniophora puteana) was investigated after long-term forest fertilization at three different sites in central Finland. Different wood properties: wood extractives, wood chemistry, and wood anatomy were used to explain sap- and heartwood decay. Scots pine sapwood was more susceptible to decay than its heartwood. In one site, sapwood seemed to be more resistant to wood decay after forest fertilization whereas the susceptibility of heartwood increased. Significant changes in the sapwood chemistry were found between treatment and sites, however, no relationship between wood chemistry and wood decay was observed in the factor analysis. The results of this study show that there was an inconsistent relationship between decay susceptibility and fertilization and the measured physical and chemical attributes of the wood were not consistently correlated with the decay rate.     

Localisation of fungal hyphae in wood using immunofluorescence labelling and confocal laser scanning microscopy

This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood.