► Thermophiles are potential candidates for efficient lignocellulose conversion. ► The limitations of existing lignocellulose conversion processes are discussed. ► Importance of thermostable enzymes ...to overcome limitations are discussed. ► Influence of high temperatures on lignocellulose conversion processes are discussed.
Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ⩽50°C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed.
The composition of thermophilic (60
°C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of ...sequences related to
Actinobacteria,
Bacteroidetes,
Chloroflexi,
Deinococcus-Thermus,
Firmicutes, and
Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera
Geobacillus,
Thermobacillus,
Cohnella, and
Thermus. A compost isolate WSUCF1 (
Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (
Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75
°C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had
K
m values of 3.11 and 1.08
mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70
°C for 1
day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.
•A gene encoding a GH10 endo-xylanase from Geobacillus sp. WSUCF1 was cloned and overexpressed.•Endo-xylanase exhibited high specific activity, activity at higher pHs and wide substrate ...specificity.•Endo-xylanase released high levels of industrially important xylo-oligosaccharides from xylan.•Better lignocellulose conversions to sugars were obtained compared to commercial endo-xylanase.
A gene encoding a GH10 endo-xylanase from Geobacillus sp. WSUCF1 was cloned and expressed in Escherichia coli. Recombinant endo-xylanase (37kDa) exhibited high specific activity of 461.0U/mg of protein. Endo-xylanase was optimally active on birchwood xylan at 70°C and pH 6.5. The endo-xylanase was found to be highly thermostable at 50 and 60°C, retaining 82% and 50% of its original activity, respectively, after 60h. High xylan conversions (92%) were obtained with oat-spelt xylan hydrolysis. Higher glucan and xylan conversions were obtained on AFEX-treated corn stover with an enzyme cocktail containing WSUCF1 endo-xylanase (71% and 47%) as compared to enzyme cocktail containing commercial fungal endo-xylanase (64% and 41%). High specific activity, active at high pH’s, wide substrate specificity, and higher hydrolytic activity on recalcitrant lignocellulose, make this endo-xylanase a suitable candidate for biofuel and bioprocess industries.
Efficient enzymatic hydrolysis of lignocellulose to fermentable sugars requires a complete repertoire of biomass deconstruction enzymes. Hemicellulases play an important role in hydrolyzing ...hemicellulose component of lignocellulose to xylooligosaccharides and xylose. Thermostable xylanases have been a focus of attention as industrially important enzymes due to their long shelf life at high temperatures. Geobacillus sp. strain WSUCF1 produced thermostable xylanase activity (crude xylanase cocktail) when grown on xylan or various inexpensive untreated and pretreated lignocellulosic biomasses such as prairie cord grass and corn stover. The optimum pH and temperature for the crude xylanase cocktail were 6.5 and 70°C, respectively. The WSUCF1 crude xylanase was found to be highly thermostable with half-lives of 18 and 12 days at 60 and 70°C, respectively. At 70°C, rates of xylan hydrolysis were also found to be better with the WSUCF1 secretome than those with commercial enzymes, i.e., for WSUCF1 crude xylanase, Cellic-HTec2, and AccelleraseXY, the percent xylan conversions were 68.9, 49.4, and 28.92, respectively. To the best of our knowledge, WSUCF1 crude xylanase cocktail is among the most thermostable xylanases produced by thermophilic Geobacillus spp. and other thermophilic microbes (optimum growth temperature ≤70°C). High thermostability, activity over wide range of temperatures, and better xylan hydrolysis than commercial enzymes make WSUCF1 crude xylanase suitable for thermophilic lignocellulose bioconversion processes.
•A method for overcoming bacterial contamination in ethanol fermentation is described.•The method utilizes beneficial microbes originally isolated as process contaminants.•This approach could ...eliminate the use of antibiotics in fuel ethanol fermentations.
Fuel ethanol fermentations are not performed under aseptic conditions and microbial contamination reduces yields and can lead to costly “stuck fermentations”. Antibiotics are commonly used to combat contaminants, but these may persist in the distillers grains co-product. Among contaminants, it is known that certain strains of lactic acid bacteria are capable of causing stuck fermentations, while other strains appear to be harmless. However, it was not previously known whether or how these strains interact one with another. In this study, more than 500 harmless strains of lactic acid bacteria were tested in a model system in combination with strains that cause stuck fermentations. Among these harmless strains, a group of beneficial strains was identified that restored ethanol production to near normal levels. Such beneficial strains may serve as an alternative approach to the use of antibiotics in fuel ethanol production.
Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and β-xylosidase. β-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. ...Thermostable β-xylosidases have been a focus of attention as industrially important enzymes due to their long shelf life and role in the relief of end-product inhibition of xylanases caused by xylo-oligosaccharides. Therefore, a highly thermostable β-xylosidase with high specific activity has significant potential in lignocellulose bioconversion.
A gene encoding a highly thermostable GH39 β-xylosidase was cloned from Geobacillus sp. strain WSUCF1 and expressed in Escherichia coli. Recombinant β-xylosidase was active over a wide range of temperatures and pH with optimum temperature of 70 °C and pH 6.5. It exhibited very high thermostability, retaining 50% activity at 70 °C after 9 days. WSUCF1 β-xylosidase is more thermostable than β-xylosidases reported from other thermophiles (growth temperature ≤ 70 °C). Specific activity was 133 U/mg when incubated with p-nitrophenyl xylopyranoside, with Km and Vmax values of 2.38 mM and 147 U/mg, respectively. SDS-PAGE analysis indicated that the recombinant enzyme had a mass of 58 kDa, but omitting heating prior to electrophoresis increased the apparent mass to 230 kDa, suggesting the enzyme exists as a tetramer. Enzyme exhibited high tolerance to xylose, retained approximately 70% of relative activity at 210 mM xylose concentration. Thin layer chromatography showed that the enzyme had potential to convert xylo-oligomers (xylobiose, triose, tetraose, and pentaose) into fermentable xylose. WSUCF1 β-xylosidase along with WSUCF1 endo-xylanase synergistically converted the xylan into fermentable xylose with more than 90% conversion.
Properties of the WSUCF1 β-xylosidase i.e. high tolerance to elevated temperatures, high specific activity, conversion of xylo-oligomers to xylose, and resistance to inhibition from xylose, make this enzyme potentially suitable for various biotechnological applications.
A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 ...demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation.
The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid, plus the generally regarded as safe status of lactobacilli, which therefore have less regulatory concerns, LfFE from the probiotic L. fermentum reported in this work can be directly used for increased production of high-value hydroxycinnamates and ferulic acid from natural or synthetic carbon sources.
Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL ...50380 on sucrose medium have a d-mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3'-O-acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d-galactitol (dulcitol), d-sorbitol (glucitol), d- and l-arabitol, d-xylitol, l-threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d-arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus. Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.
•Bacillus spp. supernatants inhibited biofilms of fuel ethanol contaminants.•Bacillus nakamurai strain NRRL B-41091 was particularly effective.•Inhibition was bacteriostatic and also affected ...planktonic cells.•Inhibition was specific for strains of Lactobacillus.•B. nakamurai prevented stuck fermentations in a corn mash model system.
Commercial fuel ethanol fermentations suffer from microbial contaminants, particularly species of Lactobacillus that may persist as antibiotic-resistant biofilms. In this study, culture supernatants from 54 strains of Bacillus known to produce lipopeptides were tested for inhibition of biofilm formation by Lactobacillus fermentum, L. plantarum, and L. brevis strains previously isolated as biofilm-forming contaminants of a commercial fuel ethanol facility. Eleven Bacillus strains inhibited biofilm formation by all three strains by at least 65% of controls. None of these strains inhibited Saccharomyces cerevisiae. Three strains also significantly inhibited planktonic cultures of Lactobacillus. Culture supernatants from B. nakamurai strain NRRL B-41091 were particularly effective. Inhibition was bacteriostatic rather than bacteriocidal, and appeared to be specific for strains of Lactobacillus. Furthermore, the inhibitor from B. nakamurai was shown to prevent stuck fermentations in a corn mash model fermentation system of S. cerevisiae contaminated with L. fermentum.
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